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ANIMAL REPRODUCTION Official journal of the Brazilian College of Animal Reproduction v.11, n.3

July/September

2014

Contents Proceedings of the 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE), August 14 to 17th, 2014, Natal, RN, Brazil.

From de SBTE President

132

From the Scientific Committee Chair

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Conferences papers Historical context of cattle embryo transfer technique in Brazil C.F.M. Rodrigues

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Large-scale chromatin structure and function changes during oogenesis: the interplay between oocyte and companion cumulus cells A.M. Luciano, F. Franciosi, C. Dieci, I. Tessaro, L. Terzaghi, S.C. Modina, V. Lodde

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Control of oocyte maturation F.C. Landim-Alvarenga, R.R.D. Maziero

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Effect of uterine environment on embryo production and fertility in cows A.H. Souza, C.D. Narciso, E.O.S. Batista, P.D. Carvalho, M.C. Wiltbank

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Effects of energy and protein nutrition in the dam on embryonic development M.C. Wiltbank, A. Garcia-Guerra, P.D. Carvalho, K.S. Hackbart, R.W. Bender, A.H. Souza, M.Z. Toledo, G.M. Baez, R.S. Surjus, R. Sartori

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Molecular markers of fertility in cattle oocytes and embryos: progress and challenges E. Orozco-Lucero, M-A. Sirard

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Nutritional status of donor cows: insulin related strategies to enhance embryo development C. Ponsart, G. Gamarra, S. Lacaze, A.A. Ponter

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Different ways to evaluate bovine sexed sperm in vitro J.O. Carvalho, R. Sartori, M.A.N. Dode

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Advances in reproductive management: pregnancy diagnosis in ruminants T.L. Ott, C. Dechow, M.L. O’Connor

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Sex-sorted sperm for artificial insemination and embryo transfer programs in cattle M.F. Sá Filho, M. Nichi, J.G. Soares, L.M. Vieira, L.F Melo, A. Ojeda, E.P. Campos Filho, A.H. Gameiro, R. Sartori, P.S. Baruselli

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The physiology and impact on fertility of the period of proestrus in lactating dairy cows M.C. Wiltbank, G.M. Baez, J.L.M. Vasconcelos, M. Pereira, A.H. Souza, R. Sartori, J.R. Pursley

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Maternal immune responses to conceptus signals during early pregnancy in ruminants T.L. Ott, M.M. Kamat, S. Vasudevan, D.H. Townson, J.L. Pate

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The role of proestrus on fertility and postovulatory uterine function in the cow M. Binelli, G. Pugliesi, V.V. Hoeck, M. Sponchiado, R.S. Ramos, M.L. Oliveira, M.R. França, F.L. D’Alexandri, F.S. Mesquita, C.M.B. Membrive

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Impact of animal health on reproduction of dairy cows J.E.P. Santos, E.S. Ribeiro

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Health and safety of IVF embryos: challenges for the international ET industry P. Blondin

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Potential practical implications of nanotechnology in animal reproductive biotechnologies L.P. Silva

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Abstracts Male Reproductive Physiology and Semen Technology (Abstracts A001 to A029)

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Folliculogenesis, Oogenesis and Superovulation (Abstracts A030 to A051)

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FTAI, FTET and AI (Abstracts A052 to A120)

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OPU-IVP and ET (Abstracts A121 to A166)

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Embriology, Biology of Development and Physiology of Reproduction (Abstracts A167 to A193)

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Cloning, Transgenesis and Stem Cells (Abstracts A194 to A201)

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Supporting Biotechnologies: Cryopreservation and Cryobiology, Image Analysis and Diagnosis, Molecular Biology and “Omics” (Abstracts A202 to A221)

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Author index to v.11, n.3, 2014

502

ANIMAL REPRODUCTION Official journal of the Brazilian College of Animal Reproduction v.11, n.3

July/September

2014

Editor-in-Chief Luiz Renato de França - UFMG, Brazil Editorial Board Andrzej Bartke - Southern Illinois University, USA Antônio Carlos S. Castro - UFMG, Brazil Arlindo A.A. Moura - UFC, Brazil Barry D. Bavister - University of New Orleans, USA Bart Gadella - Utrecht University, The Netherlands Brian Setchell - University of Adelaide, Australia Eduardo Bustos Obregón - Universidad de Chile, Chile Eduardo Paulino da Costa - UFV, Brazil Edward L. Squires - Colorado State University, USA Fernanda da Cruz Landim-Alvarenga - UNESP, Brazil George E. Seidel Jr - Colorado State University, USA Goro Yoshizaki - Tokyo University of Marine Science and Technology, Japan Heriberto Rodriguez-Martinez, SLU, Sweden Hugo P. Godinho - PUC/Minas, UFMG, Brazil João Carlos Deschamps - UFPEL, Brazil

J.A. (Lulu) Skidmore-The Camel Reproduction Centre, UAE Katrin Hinrichs - Texas A&M University, USA Keith Betteridge - University of Guelph, Canada Margaret J. Evans - CDHB, New Zealand Martha C. Gomez - Louisiana State University, USA Peter J. Broadbent - UK Rex A. Hess - University of Illinois, USA Ricardo S. Calandra - IMBICE, IBYME, Argentina Richard Fayrer-Hosken - University of Georgia, USA Rüdiger W. Schulz - Utrecht University, The Netherlands Stanley P. Leibo - University of New Orleans; Audubon Research Center, USA Sue M. McDonnell - University of Pennsylvania, USA Telma M.T. Zorn – USP, Brazil William R. Allen - University of Cambridge, UK Wilma de Grava Kempinas - UNESP, Brazil

Proof Editor: Hugo Pereira Godinho, UFMG/ICB, Belo Horizonte, MG, Brazil Proofreader: Aline Marques Kaehler Secretaries: Maria Helena Chaves da Silva, Eunice de Faria Lopes Animal Reproduction publishes reviews, original articles, and short communications related to the basic, applied and biotechnological aspects of animal reproductive biology. Manuscripts should be submitted online to the Editor-in-Chief http://www.cbra.org.br/portal/index.htm. Instructions to Authors are available at http:// http://www.cbra.org.br/portal/index.htm. Animal Reproduction (ISSN 1806-9614 printed 2004/2010; 1984-3143 online 2004-) is published quarterly and wholly owned by the Brazilian College of Animal Reproduction (Colégio Brasileiro de Reprodução Animal - CBRA). Subscriptions and renewals are based on the calendar year. Printed versions of the journal are freely available to the members of the CBRA and electronic versions are available at the journal website. All the correspondence should be sent to the following address: Editor-in-Chief, Animal Reproduction Colégio Brasileiro de Reprodução Animal - CBRA Av. Cel José Dias Bicalho 1224, Loja 04 - Bairro São José - 31275-050 Belo Horizonte, MG, Brazil. Phone: +55(31)3491-7122, Fax: +55(31)3491-7025, E-mail: [email protected], Website: http://www.cbra.org.br/portal/index.htm Animal Reproduction, v.1- , n.1- , 2004Belo Horizonte, MG, Brazil: Colégio Brasileiro de Reprodução Animal, 2004Quarterly. ISSN 1806-9614 (printed 2004/2010); 1984-3143 (online 2004-) 1. Animal reproduction - Periodicals. I. Colégio Brasileiro de Reprodução Animal. II. Brazilian College of Animal Reproduction. CDU - 636.082.4 (81) (05) - AGRIS L10

Biological Abstracts Biosis Previews CABI Abstracts Current Contents: Agric Biol Environ Sci Directory of Open Access Journals (DOAJ) Web of Knowledge CAPES/Qualis

Proceedings of the 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE), August 14 to 17, 2014, Natal, RN, Brazil. From the SBTE President Dear Colleagues, Welcome to 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE) in beautiful Natal, RN. Our main aspiration is to maintain the scientific quality that we have all became used to, while at the same time adapting to new demands of the Society. The cutting-edge science, brought by the various speakers and participants, inspiring environment of the ocean and many opportunities for scientific, social and business interactions will no doubt make it a very productive meeting. I am sure that this year’s meeting will be one of the best, because of the exciting and well balanced program, that scientific committee chair Roberto Sartori along with the members of the program committee have put together. As you know, the main focus of the Society is to fulfill both the interests and needs of its members. Then, we invite you to suggest or indicate the strengths and the weakness of the SBTE and the meeting itself to any of individual director. Your opinion is very important to us, after all, SBTE remains a member-drive society that requires your continuous input if we want to reach our full potential. As in the past, this meeting depends on the full support and involvement of our partner companies as well as our sponsors CNPq and CAPES, to whom we are deeply grateful. I also want to thank all the speakers who have agreed to attend this meeting and share their last findings with us helping to make the meeting a great success. This meeting turned in to a reality as a result of the hard work and dedication of the all SBTE Board of Directors, Roberto Sartori, Alexandre Garcia, Regivaldo Sousa, Mauricio Peixer, Mauricio Franco, Bianca Silva, Marcelo Nogueira, Ligiane Leme, José Carvalho, José Buratini, Carlos Alberto Zanenga and Osnir Watanabe. Their efforts regarding all SBTE issues have been extraordinary and they have been tireless. We hope that the ocean view and the warm reception will inspire productive interactions that will generate new research activities, new collaborations and good business. Enjoy the meeting and have fun on Natal Dunes! Margot Alves Nunes Dode, PhD President of SBTE (2014-2015)

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Proceedings of the 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE), August 14 to 17th, 2014, Natal, RN, Brazil.

From the scientific committee chair Greetings and welcome to the 28th SBTE Annual Meeting! The Scientific Committee is very happy with this new challenge, which is follow up the excellent job done by the preceding Committee. Therefore, we maintained most of the program structure for this year. We invited world-class speakers to lecture about up-to-date information on embryo technology-related fields. The novelty for this year is shorter time for presentations on the concurrent sessions on basic (science) and applied (technology) topics, but more time for discussions. We will encourage the audience to participate and engage more on debates. The opening ceremony will have the traditional talk from the IETS President, plus some history of SBTE, but from the field (Veterinary) point of view. The main program will have three plenary sessions, and six sessions: The Oocyte, The Embryo, and The Uterus (“SBTE Science”), and The Donor, Emerging Technologies, and Health (“SBTE Technology”). For the plenary sessions, speakers were carefully picked to deliver contents of general interest to the society. In the other sessions, attendees will choose between two concurrent sessions, one focused on more basic aspects (“SBTE science”) and the other emphasizing the more applied aspects (“SBTE technology”) of that session’s topic. For this year, we are innovating for the student’s competitions that will be also separated (SBTE science and SBTE technology). Special emphasis must be directed to the pre-conference workshops. We will have five workshops, with novelties. 1. Artificial Insemination (with short talks and more time for discussions); 2. Small Ruminant Reproduction (with very exciting topics); 3. The Equine Workshop that was renewed to attract more of the horse reproduction community; 4. The In Vitro Fertilization Workshop, that will discuss several topics, including International Market and Embryo Import/Export; and 5. The 1st Workshop on Functional Genomics applied to Animal Reproduction. The SBTE wants to thank all members that sent their best work to be presented at this meeting. For the poster sessions, we are also changing a bit, having presentations divided within two days. Finally, SBTE wants to acknowledge the speakers, for putting a lot of effort on the preparation of excellent manuscripts and lectures to be delivered at this year’s meeting. We also want to thank all the SBTE team and especially, the Scientific Committee and all the reviewers that worked tirelessly and enthusiastically 24/7. This list includes all folks from the SBTE administrative board, abstract session coordinators, abstract reviewers, manuscript reviewers and scientific editors. Once again, SBTE thanks the editor and staff at the Animal Reproduction journal and the Colégio Brasileiro de Reprodução Animal, for their collaborative spirit and instrumental help on putting together this year’s meeting proceedings. We hope you find this volume informative and useful. See you in Natal! Roberto Sartori, PhD Chairman of the SBTE Scientific Committee (2014-2015)

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Proceedings of the 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE), August 14 to 17th, 2014, Natal, RN, Brazil.

SBTE Executive Board (2013-2015) Margot Alves Nunes Dode (President) José Buratini Junior (Vice-president) Regivaldo Vieira de Sousa (1st Secretary) Bianca Damiani Marques Silva (2nd Secretary) Alexandre Rossetto Garcia (1st Treasurer) Maurício Machaim Franco (2nd Treasurer) Marcelo Fábio Gouveia Nogueira (Communications Director) Maurício Peixer (Business Director) José de Oliveira Carvalho (Information Technology Specialist) Osnir Yoshime Watanabe (Industry Liaison) Carlos Alberto Zanenga (Veterinary Practitioners Representative) Roberto Sartori (Scientific Committee Chair) Proceedings General Editor Roberto Sartori Scientific Editor Hugo Pereira Godinho Workshop Chairs Gustavo Férrer Carneiro Hymerson Costa Azevedo José Fernando Garcia José Luiz Moraes Vasconcelos Maurício Peixer Yeda Watanabe Awards Committee Chairs José Buratini Junior José Luiz Moraes Vasconcelos Mário Binelli Milo Charles Wiltbank Roberto Sartori Manuscript Reviewers Anneliese de Souza Traldi Fabíola Freitas de Paula Lopes Guilherme de Paula Nogueira Jo Leroy João Carlos Pinheiro Ferreira João Henrique Moreira Viana José Buratini Junior José Eduardo Portela Santos José Luiz Moraes Vasconcelos Marcel Amstalden Margot Alves Nunes Dode Maurício Machaim Franco Rafael Sisconeto Bisinotto Reuben J. Mapletoft Ricardo Chebel Roberto Sartori William W. Thatcher

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Abstract Session Coordinators Alexandre Henryli de Souza Eneiva Carla Carvalho Celeghini Felipe Perecin Fernanda da Cruz Landim José Buratini Jr. Manoel Francisco de Sá Filho Marcelo Marcondes Seneda Paula de Carvalho Papa

Abstract Reviewers Alexandre Floriani Ramos Alexandre Henryli de Souza André Furugen Cesar De Andrade Anibal Ballarotti do Nascimento Anthony Cesar Souza Castilho Antônio Chaves de Assis Neto Bernardo Gasperin Bruna de Vita Carlos Antônio Carvalho Fernandes Cezinande Meira Christina Ramires Ferreira Claudia Barbosa Fernandes Claudia Lima Verde Leal Danila Barreiro Campos Edson Guimarães Lo Turco Eneiva Carla Carvalho Celeghini Ester Caixeta Evelyn Rabelo Andrade Fabiana Bressan Fabiana de Andrade Melo Sterza Fábio Lima Felipe Perecin Felipe Zandonadi Fernanda Landin Alvarenga Flávia Lombardi Lopes Gabriel Bó Gisele Zoccal Mingoti Gláucio Lopes Guilherme de Medeiros Bastos Guilherme de Paula Nogueira Guilherme Pugliesi Gustavo Martins Gomes dos Santos Henderson Ayres Hymerson Costa Azevedo Ian Martin Inês Cristina Giometti José Antonio Dell’aqua Jr. José Buratini Jr. José Nélio S. Sales José Ricardo Figueiredo João Carlos Pinheiro Ferreira João Henrique Moreira Viana Juliano Coelho da Silveira

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Katia Cristina Silva-Santos Klibs N. Galvão Leandro Maia Letícia Zoccolaro Oliveira Ligia Pegoraro Lindsay Unno Gimenes Liza Margareth Medeiros de Carvalho Sousa Luciano Andrade Silva Luiz Sérgio Almeida Camargo Manoel Francisco de Sá Filho Marcelo Bertolini Marcelo Fábio Gouveia Nogueira Marcelo Marcondes Seneda Marcos R. Chiaratti Marcílio Nichi Maria Denise Lopes Mateus Sudano Mayra Elena Ortiz D`Avila Assumpção Mário Binelli Naiara Zoccal Saraiva Nelcio Antonio Tonizza de Carvalho Paula de Carvalho Papa Paulo Carvalho Rafael Augusto Satrapa Rafael Bisinotto Renata Simões Reno Roldi de Araújo Ricarda Maria dos Santos Ricardo Pimenta Bertolla Roberta Machado Ferreira Roberto Sartori Rodrigo de Andrade Ferrazza Rogério Ferreira Ronaldo Luis Aoki Cerri Rubens Paes de Arruda Rui Machado Sony Dimas Bicudo Thales Ricardo Rigo Barreiros Ticiano Guimarães Leite Vanda Santos Wanessa Blaschi Wilma de Grava Kempinas

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Staff Alexandre Barbieri Prata A Anaclady Aguiar Anibal Balllarotti Nascim mento Camila Spiies Jéssica Norra Drum José de Oliiveira Carvalhoo Neto Leonardo de d França e Meelo Ligiane Leeme Louise Hellen de Oliveiraa Monique Mendes M Guardiieiro Pedro Leop poldo Jerônimoo Monteiro Jr

Sponsors APES) Coordenação de Aperfeiççoamento de Peessoa do Nívell Superior (CA N de Deesenvolvimento o Científico e T Tecnológico (C CNPq) Conselho Nacional Ministério da Agriculturaa, Pecuária e Abastecimento A ((MAPA)

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Anim. Reprod., v.11, n.3, p.137-140, Jul./Sept. 2014

Historical context of cattle embryo transfer technique in Brazil C.F.M. Rodrigues1 Gertec Embriões, Bragança Paulista, SP, Brazil.

Abstract This review presents the historical context of cattle embryo transfer (ET) technique in Brazil which led to a great progress on reproductive biotechnologies turning this country into a worldwide leader on production of bovine embryos. Commercial ET in Brazil began in 1978 with the work of two pioneers, Dr. Jorge Nicolau and Walter Becker. In the 1980s the ET technique became widespread being often used by many practitioners. However, the high variation between results indicated the need of interaction with more experienced technicians from abroad and with Brazilian researchers. Originally, there were regulatory restrictions on trade of hormones for superovulation, as well as low treatment response. In 1986, the first Brazilian Embryo Transfer Society (SBTE) meeting, held in Jaboticabal-SP, encouraged the search for better results. The goal of this meeting was to join distinguished researchers in the upcoming area of reproductive biotechnology with veterinary practitioners. This exchange was a success, leading to an outstanding utilization of reproductive biotechnologies by the Brazilian technicians. As a result, Brazil turned into a reference country in this technology. In 1985, the first Brazilian calf derived from frozen embryo was born in Sertãozinho, SP. This was followed by an improvement on embryo holding, freezing/thawing protocols. There are challenges, but it is irrefutable that we have refined the technology of embryo manipulation and genetic improvement. Those achievements are due mainly to the closer relationship between the academy, through its researchers, and the veterinary practitioners. Working together is the most efficient way to provide an outstanding environment for reproductive biotechnology innovation in Brazil. Keywords: development, embryo transfer, techniques. History The first commercial bovine embryo transfer (ET) set up in Brazil was established in the seventies. In 1978, two contemporary and independent attempts of non-surgical embryo collections followed by surgical transfers were performed, both using Bos taurus donors with fresh embryos transferred to synchronized recipients. These two pioneers, Dr. Jorge Nicolau _________________________________________ 1 Corresponding author: [email protected] Phone/Fax: +55(11)4034-0819 Received: May 27, 2014 Accepted: July 16, 2014

(DMV) at Fazenda São Pedro, Sorocaba, SP, and Dr. Walter A. P. Becker transferred fresh embryos. A year later, Dr. Aurelino Menarin Jr., in collaboration with the University of Colorado, initiated the use of ET at Campo Verde Farm in Senhor do Bonfim, Bahia, resulting in the birth of five Nelore calves. This was the earliest ET report in this breed. The 80s were considered as a landmark in expansion and establishment of ET in Brazil. More and more veterinarians were interested in applying ET in their routine. However, initial results were still variable and inconsistent which indicated the need for further research. Moreover, the knowledge exchanged with professionals from other countries was extremely helpful in order to dissipate possible doubts regarding technical issues. These, were the key factors for the maximization of the reproductive potential of donors with high genetic value. Superovulation programs aiming to increase the number of viable structures with high probability to generate pregnancy were initially performed using pregnant mare serum gonadotrophin (PMSG; Maturon Laboratório Organon do Brasil). The work developed by Elsden el al. (1976) indicated that a combination of luteinizing and follicle stimulating hormones (FSH/LH; FSH-P; Burns- Biotec Laboratories Inc., Omaha, NE) generated a greater number of viable embryos as compared to PMSG. However, this hormonal combination was not commercially available in Brazil. Therefore, most of the superovulations in those days were performed using human menopausal gonadotropin (hMG), product with equal amounts of FSH and LH, available for human. Nevertheless, results were still inconsistent. Among the limitations of superovulation hormonal programs, one can highlight the high cost of PMSG therapy. This drug was commercially available for human, and was expensive at the dose required for bovine. The hormone FSH-P, produced in the United States, was not regulated in Brazil. Some of these reagents were blocked in the airports customs leading to significant economic losses to several field professionals with works already in progress. In 1995, FSH-P was no longer produced. In the late 80s and early 90s, the imports of two commercial FSH-based products were regulated, Foltropin-V (Vetrepharm, London, Ontario) and Pluset (Hertape-Calier), allowing the intensification of studies on bovine superovulation.

Rodrigues. Historical aspects of embryo techniques in Brazil.

After the standardization on the use and dosage of both products, results became more consistent and productive. In this context, studies began to focus on embryo cryopreservation aiming to improve the logistic of ET and to create cryopreserved embryo banks. In 1985, after several attempts, the first Nelore calf originated from a cryopreserved embryo was born due to the efforts of the technical staff of Lagoa da Serra, using 10% of Glicerol (1.4 M) and decreasing concentrations of glycerol (0.7, 0.5 and 0.25 M) during the dehydration and rehydration (Leibo and Mazur, 1978). Considering that the final goal was to enable embryo trade similar to that routinely used for semen, it was important to simplify the processes of freezing, thawing and non-surgical transfer. Therefore, in 1985 Lagoa da Serra invested in a one-step technique of embryo cryopreservation/transfer (Leibo, 1984). This technique consisted in cryopreserving embryos in straws containing both glycerol and sucrose. Embryos were cryopreserved in glycerol 10% and, while filling the straw with the dehydrated embryo, two columns of sucrose, extracellular cryoprotectants, were added to the extremities of the straw. During thawing, the columns (embryo + 10 glycerol, sucrose) were gently homogenized; the embryo in contact with the sucrose would initiate the rehydration process. After 10 min, embryos were then transferred to previously synchronized recipients. However, while encouraging results were obtained with Bos taurus embryos, the same could not be observed in Bos indicus embryos. In July 1986, the first annual Brazilian Embryo Technology Society (SBTE) meeting held at the Faculdade de Ciências Agrárias e Veterinárias (FCAVUNESP), in Jaboticabal, SP, demonstrated that embryo transfer biotechnology was already a reality in Brazil. Several studies were presented by distinguished researchers, sharing questions regarding their ET field trials. Researchers such as Roberto A. de Bem, Luiz Eustáquio L. Pinheiro, José Luiz Rodrigues, Cesar R. Esper, Ricardo M. Gregory, Joaquim Mansano Garcia among others, together with experienced field professional such as Jorge Nicolau, Roberto Jorge Chebel, Carlos Fernando Marins Rodrigues, Douglas B Gaetti and Teodoro Vaske, discussed issues that, at that moment, would dominate the future application of the technique. An interesting example was the in vitro maturation of bovine embryos, project developed by the team led by Prof. Joaquim Mansano Garcia (Garcia et al., 1986). At the end of the 80s, sophisticated procedures were incorporated to the ET routine such as the embryo bipartition technique. Several research groups dedicated their time to this technolog allowing the production of identical twins and sex determination through embryo micromanipulation (Lopes et al., 2001). In the early 90s, standardization of ultrasound procedure for reproductive purposes (Kastelic et al., 138

1990) allowed determination of fetal gender (Curran, 1992), embryo and fetal development, embryo loss and, follicular dynamics leading to the development of new knowledge, new paths, new technologies and protocols. In this scenario, Brazil began to master the technique of in vitro fertilization (IVF). In 1993, a group led by Prof. Enoch Borges de Oliveira, from FCAV-UNESP, Jaboticabal, produced the first calf by IVF in Brazil. Oocytes used in this study were collected from ovaries obtained from slaughterhouse cows. Based on studies developed in Canada by Dr. Roger Sauve, in 1995, efforts were made to obtain oocytes by ultrasound guided follicular aspiration. This resulted in the birth Brazilian commercial product using this technique in 1997. In 1996, Gertec Embriões, Beabisa Agricultura and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) supported the first commercial IVF laboratory (Rodrigues and Garcia, 2000) at Fazenda Suíço, Pitangueiras, SP. This project was coordinated by Prof. Joaquim Mansano Garcia at FCAV-UNESP Jaboticabal. Doctors André Dayan and Yeda Watanabe also participated in such effort. They would later found the Vitrogen in 1998, which was a determining factor for growth of the field application of IVF and ET in Brazil. The establishment of ET and IVF in Brazil (Viana et al., 2012) has been related to several factors such as the high potential of Zebu breeds as oocyte donors, considerably superior than European breeds. Also, the fact that hormonal treatment and superovulation protocols were not required to obtain sustainable pregnancies, together with the possibility of using aged females or those that no longer respond to superovulation protocols, allowed the growth of these reproductive technologies. The advantages were also clear due to the availability of recipient females, creation of genetic improvement programs, the valuing of top animals in auctions and the leadership on beef exportations. These facts also increased the number of professionals providing such service. This scenario contributed to the Brazilian world leadership on IVP, especially in Bos indicus breeds. In this context, the use of sexed semen allowed an impressive growth on IVP. In 2011, 318,000 IVP embryos were produced in contrast to 35,563 embryos produced by superovulation. In the next year, Brazil had 86% of the IVP embryos produced worldwide (International Embryo Transfer Society - IETS, 2013). Final considerations What are the perspectives for the Brazilian livestock genetic market? Currently, the biotechnological market trends highlight the importance of dairy breeds, with almost 70% of workload of field professionals, especially for Gir, Girolando, Holstein and Jersey breeds. Anim. Reprod., v.11, n.3, p.137-140, Jul./Sept. 2014

Rodrigues. Historical aspects of embryo techniques in Brazil.

Brazil is the world supplier of Zebu genetic material. What are the reasons? Several factors are responsible for such scenario: 1) Brazil has the largest commercial Zebu herd in the world; 2) Highly capacitated Artificial Insemination and Embryo Transfer Centrals and in vitro fertilization laboratories; 3) Governmental and Class programs for Genetic Improvement (EMBRAPA, ABCZ, USP); 4) Standardized distribution of agricultural inputs; 5) Academic and technical infrastructure; 6) Biosafety of the exported products; and 7) National tropical technology and experience. What are the reasons for the stagnation of the exports? 1) International prejudice and unawareness regarding the eradication of serious diseases such as foot and mouth disease in Brazil; 2) Brazil was listed as level 1 (low risk) on sanitary issues by the World Organisation for Animal Health (OIE), as stated on the OIE 80th General Session; 3) Sanitary protocols that are not practical and not viable (e.g., a protocol recently signed with Costa Rica) from the technical to the economical perspectives; 4) Lack of a strategic planning involving all the governmental levels. Several Ministries must be involved in a common objective since problems are related to logistic, commercial and income politics, institutional issues and compliance with current legislations. In 2013 the semen market traded 13 million straws. This represents an increase of 5.4% in comparison with 2012 (Associação Brasileira de Inseminação Artificial - ASBIA, 2013). This is especially due to the increase on sales of dairy breeds which increased from 4.9 to 5.3 million (9.6%). Even on beef breeds, an increase could be observed from, 7.4 to 7.6 million of straws traded. Despite the reduction when compared to the year before, participation of beef breeds on sales are still the majority when compared to the dairy breed. The later, however, showed an increase from 39.6% in 2012 to 41.2% in 2013. A cause for apprehension is the drop on the sale of Nelore breed semen straws, with 11.74% on Nelore Padrão and 15.13% on Nelore Mocho. Leadership belongs now to the Angus breed with 44% of the beef market, with the Aberdeen Angus being responsible for a growth of 24.5% when comparing to 2012. The importance of Zebu breeds for countries located in the tropics justifies such concern since investments are made with breeding programs and testing of highly reliable bulls. Because the investments on Angus breed are originated mostly on imports, such increase in this breed indicates economic losses to the country’s commercial balance. This increase occurred due to the increase of timed artificial insemination (TAI) in combination with the implementation of Bos taurus/Bos indicus cross breeding, aiming to produce meat with higher quality and consequent valuing of the final product. Anim. Reprod., v.11, n.3, p.137-140, Jul./Sept. 2014

Embryo market has become a profitable activity worldwide, especially after the advantages provided by embryo cryopreservation. The interest of countries located in the tropics for dairy Zebu genetics has increased since 2005. This is mainly due to the continuous work of genetic improvement and bulls’ fertility and progeny testing developed by Embrapa, ABCZ and USP. As an example, the Girolando breed that contributes to 68% of the dairy animals in Brazil, is responsible for 80% of the milk produced. The Genetic Improvement Program in this breed started 17 years ago with the technical coordination of EMBRAPA Gado de Leite. Countries such as Panama, Costa Rica, Paraguay, Colombia, Bolivia, and Canada, have invested in the Brazilian genetics. However, our participation is still incipient, especially due to unfeasible sanitary protocols, both on technical and economic aspects. The development of new technologies is essential and fundamental to the growth and improvement of the existing animal genetics in our country. Biotechnologies such as cloning, transgenesis, animal selection based on molecular markers, all developed to improve the participation in a highly competitive market, still present a reduced commercial efficiency. However, we believe that this should be the current approach since reproductive biotechnologies have shown to increase the efficiency of genetic selection. These previous advances have occurred due to the pioneering efforts of two groups that worked together. On one side, researchers, developers of technical procedures and embryo production techniques; on the other side, field veterinarians, modifiers and adapters of techniques aiming to engage with the commercial reality of the rural producers. References Associação Brasileira de Inseminação Artificial (ASBIA). 2013. Índex ASBIA: Importação, Exportação e Comercialização de Sêmen. 2013. Available on: http://www.asbia.org.br/. Accessed on: July 5, 2014. Curran S. 1992. Fetal sex determination in cattle and horses by ultrasonography. Theriogenology 37:17-20. Elsden RP, Hasler JF, Seidel GE Jr. 1976. Non surgical recovery of bovine eggs. Theriogenology, 6:523-632. Garcia JM, Pinheiro LEL, Mikich AB, Lima VF. 1986. Manutenção in vitro de oocitos bovinos cultivados em meios quimicamente definidos e gonadotrofinas. In: Anais da I Reunião Anual da Sociedade Brasileira de Transferencia de Embriões, 1986, Jaboticabal, SP. Jaboticabal, SP: SBTE. International Embryo Transfer Society (IETS). 2013. Embryo Transfer Newsletter. Available on:http//www.iets@org. Accessed on: June 1st, 2014. Kastelic JP, Pierson RA, Ginther OJ. 1990. 139

Rodrigues. Historical aspects of embryo techniques in Brazil.

Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers. Theriogenology, 34:487-498. Leibo SP, Mazur P. 1978. Methods for the preservation of mammalian embryos by freezing. In: Daniel Jr JC (Ed.). Methods in Mammalian Reproduction. New York, NY: Academic Press. pp. 179-201. Leibo SP. 1984. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos.

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Theriogenology, 21:767-790. Lopes RF, Forell F, Oliveira AT, Rodrigues JL. 2001. Splitting and biopsy for bovine embryo sexing under field conditions. Theriogenology, 56:1383-1392. Rodrigues CFM, Garcia JM. 2000. Fecundação in vitro em bovinos: aplicação comercial. Arq Fac Vet UFRGS, 28(suppl):186-187. Viana JHM, Siqueira LGB, Palhao MP, Camargo LSA. 2012. Features and perspectives of the Brazilian in vitro embryo industry. Anim Reprod, 9:12-18.

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Large-scale chromatin structure and function changes during oogenesis: the interplay between oocyte and companion cumulus cells A.M. Luciano1, F. Franciosi, C. Dieci, I. Tessaro, L. Terzaghi, S.C. Modina, V. Lodde Reproductive and Developmental Biology Laboratory, Department of Health, Animal Science and Food Safety, University of Milan, Milan, Italy.

Abstract The process of chromatin configuration remodeling within the mammalian oocyte nucleus or germinal vesicle (GV), which occurs towards the end of its differentiation phase before meiotic resumption, has received much attention and has been studied in several mammals. This review is aimed to highlight the relationship between changes in chromatin configurations and to both functional and structural modifications occurring in the oocyte nuclear compartment. During the extensive phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. Then the decondensed chromatin undergoes profound rearrangements during the final stages of oocyte growth in tight association with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional crosstalk with the follicular cells, experimental manipulation of large-scale chromatin configuration is discussed. Besides providing tools to determine the key cellular pathways involved in genome-wide chromatin modifications, the present findings will aid to the refinement of physiological culture systems that can have important implications in treating human infertility as well as managing breeding schemes in animal husbandry. Keywords: chromatin, cumulus cells, gap junctions, germinal vesicle, oocyte, transcriptional activity. Introduction The chromatin organization and architecture is a characteristic element of the process of oocyte differentiation in mammals (Luciano and Lodde, 2013). Oocyte development is characterized by impressive _________________________________________ 1 Corresponding author: [email protected] Phone: +39(02)5031-7969 Received: May 13, 2014 Accepted: July 1, 2014

changes in chromatin structure and function within the nucleus, namely the germinal vesicle (GV). These changes are crucial to confer the oocyte with meiotic and developmental competences and they occur along the process of folliculogenesis, when gamete and somatic cells communicate through junctional and paracrine mediated mechanisms (Albertini et al., 2003). Dynamic changes in GV oocyte chromatin configuration have been described in mouse (Wickramasinghe et al., 1991; Debey et al., 1993; Zuccotti et al., 1995), rat (Mandl, 1962), human (Combelles et al., 2003; Miyara et al., 2003), monkey (Schramm et al., 1993), horse (Hinrichs and Williams, 1997; Hinrichs and Schmidt 2000; Franciosi et al., 2012), pig (Bui et al., 2007; Dieci et al., 2013), cattle (Fuhrer et al., 1989; Chohan and Hunter, 2003; Liu et al., 2006; Lodde et al., 2007), buffalo (Yousaf and Chohan, 2003), goat (Sui et al., 2005), sheep (Russo et al., 2007), dog (Jin et al., 2006; Lee et al., 2008; Reynaud et al., 2009), ferret (Sun et al., 2009), rabbit (Wang et al., 2009) and cat (Comizzoli et al., 2011). Although different patterns of chromatin organization have been defined in mammals, sometimes the nomenclature can be confusing, since it is not univocal in part due to some species-specificity. For example, Surrounded Nucleolus (SN) configuration - where chromatin forms a ring around the nucleolus - has been described in the mouse as well as and in other mammals (monkey, pig, rat and human) while this configuration was not evidenced in the horse oocyte where ‘fibrillar’, ‘intermediate’ and ‘condensed’ configurations were documented (Franciosi et al., 2012), or in the bovine, where the highest degree of chromatin compaction is found in GV3 oocytes. Moreover, very often, different acronyms were used within the same species by different authors and this made data interpretation puzzling. Nevertheless, despite the species-specific patterning, the process of large-scale chromatin configuration changes seems to be a common process in mammals. In fact, what is clear is that the chromatin contained in the GV achieves a high degree of condensation and compaction passing through intermediate configurations, before the resumption of meiosis. Incidentally, it is worth stating that the GV3 or the SN configurations have been first described by Blackman in early 1900 in spermatocytes of millipedes

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named the ‘karyosphere’ (Blackman, 1903). The karyosphere “represents a transformation of meiotic chromosomes often occurring just prior to the completion of meiotic division”, and a similar structure, named karyosome, exists in Drosophila (King, 1970) as well as in other phylogenetically distant organisms studied so far, suggesting a well-conserved process between species during phylogeny (Gruzova and Parfenov, 1993). Significance of large-scale chromatin configuration changes Differences in chromatin configuration do not only refer to morphological modifications but also to its functionality (De La Fuente, 2006; Luciano and Lodde, 2013). Several studies indicated that there is a relationship between chromatin configurations, transcriptional activity, epigenetic signature, characteristics of the ooplasm and oocyte competence and altogether these features are strictly associated one to each other. Importantly, a direct relationship between oocyte chromatin configuration and embryonic developmental competence has been ascertained in mouse (Zuccotti et al., 1998, 2002) and in cow (Lodde et al., 2007; Luciano et al., 2011). In growing mouse oocytes chromatin is initially decondensed in a configuration termed NonSurrounded Nucleolus (NSN; Mattson and Albertini 1990; Debey et al., 1993; Zuccotti et al., 1995). With the subsequent growth and differentiation, chromatin becomes progressively condensed, forming a heterochromatin rim in close apposition with the nucleolus, acquiring a configuration termed Surrounded Nucleolus (SN; Mattson and Albertini 1990; Debey et al., 1993; Zuccotti et al., 1995). The morphological variances between these two types of oocytes have a biological relevance because NSN and SN configurations have been correlated with differences in follicle size, oocyte diameter and the age of the mouse (Mattson and Albertini 1990; Zuccotti et al., 1995, 1998). It has been demonstrated that the transition into the SN configuration correlates with the timely progression of meiotic maturation (Wickramasinghe et al., 1991; Debey et al., 1993; Zuccotti et al., 1995) suggesting that SN oocytes may represent the more advanced stage of preovulatory oocytes (Mattson and Albertini 1990; Zuccotti et al., 1995, 1998). Additionally, after in vitro maturation and fertilization, NSN oocytes are unable of development beyond the two-cell stage while SN oocytes are capable of development to the blastocyst stage (Zuccotti et al., 1998, 2002). Differences in chromatin configurations have also been correlated with changes in transcriptional activity, with NSN oocytes transcriptionally active and SN oocytes associated with global repression of transcriptional activity (Bouniol-Baly et al., 1999; Christians et al., 1999; De La Fuente and Eppig 2001; 142

Liu and Aoki, 2002; Miyara et al., 2003). In the cow, oocytes collected from early and middle antral follicles present four patterns of chromatin configuration (Fig. 1), from GV0 to GV3 characterized by progressive increase in condensation (Lodde et al., 2007), transcriptional silencing (Lodde et al., 2008; Luciano et al., 2011), global DNA methylation (Lodde et al., 2009) and progressive histone H4 acetylation (unpublished data), as previously reported also in mice (Akiyama et al., 2004). As shown in Fig. 2, the GV0 stage shows a diffuse filamentous pattern of chromatin in the whole nuclear area; the GV1 and GV2 configurations represent early and intermediate stages, respectively, of chromatin remodeling, a process starting with the appearance of few foci of condensation in GV1 oocytes and proceeding with the formation of distinct clumps of condensed chromatin in GV2 oocytes; the GV3 is the stage where the highest level of condensation is reached with chromatin organized into a single clump (Lodde et al., 2007). Importantly, oocytes with a GV0 configuration showed a very limited capacity to resume and complete meiosis I after in vitro maturation, while virtually all the GV1, GV2 and GV3 oocytes were able to reach MII stage, despite their GV configuration. On the contrary, only a limited percentage of GV1 oocytes reached the blastocyst stage after in vitro fertilization, while GV2 and GV3 oocytes showed a higher embryonic developmental potential (Lodde et al., 2007). These results further support the general principle that meiotic and developmental competencies are acquired at sequential stages of oogenesis (Albertini et al., 2003), concomitantly with changes in large-scale chromatin structure (De La Fuente, 2006) and that chromatin remodeling can be considered a marker of oocyte differentiation and developmental competence. The progressive large scale chromatin remodeling relies on functional gap-junction mediated communications between oocyte and follicular cells During folliculogenesis oocyte growth and differentiation tightly depend on the establishment of a patent bidirectional communication between oocytes and companion granulosa cells mediated by heterologous gap junctions (Eppig, 2001; Matzuk et al., 2002; Mehlmann et al., 2004). In mouse, previous studies indicate that the presence of oocyte-associated granulosa cells are required for the progressive repression of transcriptional activity in fully grown oocytes (De La Fuente and Eppig, 2001) and to promote the transition from NSN to SN configuration after gonadotropin stimulation (De La Fuente and Eppig, 2001). This hypothesis is supported also by studies where gap junction mediated communications (GJC) between mouse oocyte and cumulus cells were interrupted, due to targeted deletion of the connexin 37 gene (Gja4), and chromatin condensation associated Anim. Reprod., v.11, n.3, p.141-149, Jul./Sept. 2014

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with transcriptional repression failed to occur (Carabatsos et al., 2000). Coupling between oocyte and cumulus cells undergoes dynamic changes during follicle development and the patency of GJC between the two compartments decreases in parallel with the meiotic resumption of the oocyte (Eppig, 1982; Larsen et al., 1986, 1987). However, recent studies performed in the cow, horse, dog, cat and pig (Luvoni et al., 2001, 2006; Colleoni et al., 2004; Luciano et al., 2004; Dieci et al., 2013) indicated that morphologically healthy oocyte-cumulus cells complexes isolated from antral follicles without evident signs of atresia form a heterogeneous population characterized by different degree of GJC functionality. In the cow, the direct oocyte-granulosa cell communication through gap junctions seems a requisite for chromatin remodeling during the final phase of oocyte growth (Lodde et al., 2007; Luciano et al., 2011). This is supported by the evidences that, at the time of collection, the pattern of uncondensed chromatin in GV0 oocytes is associated with fully open GJC. On the contrary, the percentage of oocytes with functionally open communications significantly decreases with the increase of chromatin condensation, from GV1 to GV3 oocytes (Lodde et al., 2007; Luciano et al., 2011), indicating that when oocytes reach the highest level of chromatin condensation, there is a greater probability of loosing coupling with follicular cells (Lodde et al., 2007). On the other hand, the increase in chromatin condensation may represent a consequence of the premature interruption of the communication between the oocyte and follicular cells before final oocyte maturation, since the loss of GJC between the germ and somatic compartment has been related with early events of follicular atresia (Wiesen and Midgley, 1993). The manipulation of GJC functionality affects chromatin configuration and transcription through cAMP-mediated mechanism(s) The central role of GJC in the modulation of chromatin configuration, global transcriptional activity and developmental competence acquisition has been recently confirmed in bovine oocyte-cumulus cells complexes. The use of culture systems that prolonged GJC functionality sustained oocyte growth and permitted chromatin to gradually organize from GV0 to the GV1 configuration, thus allowing the oocyte to acquire the ability to mature and to be fertilized in vitro (Luciano et al., 2011). .Yet, when GJ functionality was experimentally interrupted with the uncoupler 1heptanol, chromatin rapidly condensed and RNA synthesis suddenly ceased. Interestingly, this effect was nullified by treatment with cilostamide, a specific inhibitor of the oocyte-specific PDE3, an enzymedegrading cAMP (Richard et al., 2001; Conti et al., 2002; Sasseville et al., 2009), indicating that the functional status of GJC may affect both transcriptional Anim. Reprod., v.11, n.3, p.141-149, Jul./Sept. 2014

activity and remodeling of large-scale chromatin configuration, potentially through cAMP-dependent mechanism(s; Luciano et al., 2011). Therefore, besides the well-characterized mechanisms of action by which cAMP is known to regulate meiotic resumption (Downs, 2010; reviewed in Bilodeau-Goeseels, 2011), these studies may suggest that cAMP could be also involved in controlling the activity of factors that modulate transcription and largescale chromatin remodeling during the final phase of oocyte growth and before the resumption of meiosis. In fact, since the preservation of a proper cAMP content in the oocyte even in the absence of functional GJC is able to prevent the abrupt condensation of the chromatin this makes cAMP the molecule that mostly mediates GJ action on the chromatin. Oocyte cAMP levels are sustained by endogenous adenylate cyclases and constitutively active G-protein-coupled receptors (Mehlmann et al., 2002). cAMP is generated also by cumulus cells and then transported into the oocyte through gap junctions (Anderson and Albertini 1976; Bornslaeger and Schultz, 1985). The manipulation of intracellular cAMP concentration has been demonstrated to influence functional coupling between oocyte and cumulus cells; a decrease in cAMP was accompanied by a drop in functional coupling (Luciano et al., 2004; Thomas et al., 2004). Several attempts have been made in order to mimic the physiological system in oocyte in vitro maturation taking into account the time for completing the developmental competence acquisition. These culture systems (namely pre-maturation systems) that precede in vitro maturation (Gilchrist and Thompson, 2007; Gilchrist, 2011; reviewed by Bilodeau-Goeseels, 2012) are based on the control of spontaneous meiosis resumption through the addition of either cAMP analogues or adenylate cyclase activator, PDE inhibitors (general or specific), or through a combination of these treatments. These treatments prevent the loss of cumulus-oocyte GJ mediated communications and increase oocyte developmental competence (Luciano et al., 1999; Guixue et al., 2001; Atef et al., 2005; Nogueira et al., 2006; Ozawa et al., 2008; Shu et al., 2008; Nogueira and Vanhoutte, 2009; Albuz et al., 2010; Luciano et al., 2011; Dieci et al., 2013; Lodde et al., 2013; Rose et al., 2013; Zeng et al., 2013; Richani et al., 2014). In several systems, the maintenance of a proper cAMP concentration seems to be the main requirement to promote regular chromatin transition thus endorsing oocyte differentiation (Vanhoutte et al., 2007; Luciano et al., 2011; Dieci et al., 2013; Lodde et al., 2013). Chromatin manipulation in assisted reproduction technologies There is no doubt that the experimental manipulation of large-scale chromatin configuration in 143

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vivo and in vitro will provide a tool to determine the key cellular pathways and oocyte-derived factors involved in genome-wide chromatin modifications. However, assessment of large-scale chromatin configurations has also key implications in ARTs both in human and domestic mammals. It has been shown that different patterns of chromatin configuration are indicative of different metabolic properties, thus potentially representing a morphological marker to select a population of oocytes with different cultural requirements. Several studies support the notion that in vitro treatments aiming to improve the developmental capability of immature oocytes can have a different outcome with pre-maturation culture depending on the metabolic status of the oocyte at the time of its removal from the follicular environment (Nogueira et al., 2006; Vanhoutte et al., 2008, 2009). This has been confirmed also by morphological studies in the cow, which demonstrated that the pharmacological pre-maturation system can negatively affect oocytes obtained from medium antral follicles when compared with those isolated from earlier stages (Fair et al., 2002). It is of extreme importance to realize that attempts to manipulate in vitro large-scale chromatin configuration must be performed cautiously. In fact, even though it is true that the chromatin configuration of an oocyte is indicative of its developmental capability

at the time of its collection from the follicle, pharmacological treatments forcing chromatin abruptly into a high-condensed state may not necessarily be beneficial to the oocyte competence, although fundamental in basic science-type investigation (Comizzoli et al., 2011). Therefore, the design of prematuration strategies must take into account that chromatin condensation and spatial reorganization should occur gradually and orderly, recapitulating the process that normally occurs in vivo. For example, maintenance of a proper functional coupling between oocyte and cumulus seems to be crucial in sustaining an orderly chromatin condensation in vitro (Luciano et al., 2011; Dieci et al., 2013; Lodde et al., 2013; Franciosi et al., 2014, Reproductive and Developmental Biology Laboratory, University of Milan, Milan, Italy, unpublished data). Thus, if coupling is prematurely interrupted - i.e., when oocytes have not yet acquired full competence and are still committed to accumulating transcripts and proteins - unexpected chromatin condensation can be triggered, thus preventing proper and gradual differentiation of large-scale chromatin configuration and function. In view of all given considerations, knowledge of the molecular mechanism(s) leading the oocyte to remodel its chromatin configuration under physiological conditions will be of great help for assisted reproductive technologies.

Figure 1. Transcriptional activity, global methylation, histone H4 acetylation, meiotic and developmental competence in relation to chromatin configuration in the bovine oocyte.

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Figure 2. Bright field and fluorescent images after Hoechst 33342 labeling of bovine oocytes with GV0 (A, A1), GV1 (B, B1), GV2 (C, C1), and GV3 (D, D1) configuration (see text for stage definitions). Arrows in the bright fields indicate the nuclear envelope. Scale bar: 50 mm. From: Lodde et al., 2007.

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Control of oocyte maturation F.C. Landim-Alvarenga1, R.R.D. Maziero Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, SP, Brazil.

Abstract Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte. Keywords: adenyl cyclase, cAMP, cumulus-oocytecomplex, meiosis, phosphodiesterase. Introduction The ovaries of neonate females contain oocytes in the diplotene stage of first meiotic prophase. These immature oocytes remain in a resting phase known as _________________________________________ 1 Corresponding author: [email protected] Phone/Fax: +55(14)3880-2124. Received: May 22, 2014 Accepted: July 11, 2014

dyctiate or germinal vesicle (GV) stage (Downs, 1993; Sathananthan, 1994). Within the follicle, granulosa cells (GC) that surround the oocyte undergo differentiation forming the cumulus-oocyte-complex (COC). A well-organized zona pellucida separates the oocyte from the cumulus cells (CC). The CC in close contact with the zona pellucida, which is known as cumulus oophorus (CO), communicates with the oocyte through gap junctions. The CC including the CO cells, due to their close contact with the oocyte, have a different function from that of GC from the follicular wall. Regulatory substances produced by the oocyte have important functions on the metabolism of CC and products of these somatic cells actively participate in the growth and maturation of oocytes. At puberty some of the activated follicles continue to develop, eventually resulting in ovulation. Through activation and development of the follicle, the oocyte also starts to grow. During this time, it is very active with intense mRNA and protein synthesis and differentiation of organelles. These changes prepare the oocyte to be a competent gamete (Cran and Moor, 1990). The diameter of the oocyte also increases five times (attaining 100 to 120 µm in diameter), but it still remains at germinal vesicle stage of meiosis (Schultz et al., 1978; Downs, 1993). The final process of oocyte maturation consists of the acquisition of the capacity to be fertilized and is characterized by several changes including the resumption of meiosis (Paynton and Bachvarova, 1990; Eppig, 1991). In vivo oocyte maturation coincides with differentiation of the pre-ovulatory follicle which includes changes in the oocyte and the CCs. The changes in the oocyte occur sequentially and synchronously under the stimulus of the LH, through its action on the GCs (Cran and Moor, 1990; Sathananthan, 1994). During oocyte maturation in preparation for fertilization and subsequent embryo development, changes in the nucleus and cytoplasm occur. As meiosis proceeds chromosomes segregate and organelles reorganize in the cytoplasm, transcription ceases, the stored mRNA is partially used, and the pattern of protein phosphorylation changes (Ponderato et al., 2001; Ferreira et al., 2009). Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles (Edwards et al., 1965), cytoplasmic maturation

Landim-Alvarenga and Maziero. Oocyte maturation.

seems to occur asynchronously with nuclear maturation (Janssenswillen et al., 1995; Huang et al., 1999). This is probably the main factor responsible for the lower rates of embryo production when oocytes are matured in vitro. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte. Control of meiotic arrest at the germinal vesicle stage Although the exact mechanism of oocyte maturation in vivo or in vitro is not clearly understood, it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic arrest in mammalian oocytes (Conti et al., 2012). An increased level of cAMP in the oocyte is essential for the maintenance of the blockage of meiosis, while a decrease in concentration of cAMP allows the resumption of meiosis (Sela-Abramovich et al., 2006; Conti et al., 2012). The concentration of cAMP in the oocyte is regulated by an equilibrium between two enzymes: adenyl cyclase (AC) and the phosphodiesterase (PDE), which are responsible for the synthesis and degradation,

LHR

GPR

respectively, of cAMP (Conti et al., 2002). The production of cAMP in the oocyte is controlled by receptors linked to the G protein (GPR3 in mouse) which is essential for the activation of adenyl cyclase (Holt et al., 2013). Since meiotic arrest at the germinal vesicle stage depends upon the interaction between the oocyte and surrounding CC, it has been hypothesized that cAMP passes from these cells to the oocyte through the gap junctions (Dekel et al., 1988). Besides, at least in rodents, GC play an important role in increasing cAMP in oocytes through the transfer of cyclic guanosine monophosphate (cGMP), an inhibitor of phosphodiesterase 3 (PDE3) through the gap junctions (Zhang et al., 2010). In rodents, cGMP is synthesized by CC under the influence of natriuretic peptide type C (NPPC) from the mural GC (Fig. 1; Holt et al., 2013). The effect of the cGMP in the CC is also mediated by the regulation of others phosphodiesterases (PDE2, PDE4 and PDE5; Zhang et al., 2010). High levels of cAMP in the oocyte suppress the activity of the maturation promoting factor (MPF) through a mechanism which depends on protein kinase A (PKA; Maller, 1980; Bornslaeger et al., 1986). The MPF is a protein composed of a catalytic subunit, the cycline-dependent kinase 1 (CDK1) and a regulatory subunit, the cycline B (Downs, 1993; Taieb et al., 1997), and is regulated by the phosphorylation of the treonine 14 and tyrosine 15 residues of CDK1 (Bilodeau-Goeseels, 2012). This phosphorylation is catalyzed by the kinase Wee1B, while the dephosphorylation is dependent on the phosphatase Cdc25 (Lew and Kornbluth, 1996).

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Figure 1. Schematic representation of meiotic arrest when the oocyte nucleus is at the germinal vesicle stage. GC= mural granulosa cells; CC= cumulus’cells; Oo= oocyte; PDE= phosphodiesterase; GPR= receptor linked to protein G; NPPC= natiuretic peptide; Cx= conexin. Adapted from Conti et al., 2012.

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The inhibition of PDE3 increases the levels of cAMP activating protein kinase (PKA). The PKA regulates the activity of Wee1B and Cdc25 directly. In oocytes arrested in meiotic prophase, the PKA mediates the phosphorylation of Cdc25 down-regulating its function through sequestration to the cytoplasm (Zhang et al., 2008; Pirino et al., 2009). On the other hand, the phosphorylation of Wee1B increases the inhibition of MPF. The prevention of premature resumption of meiosis is crucial for oocyte survival. In addition, the regulation of cyclin B1 levels is a second mechanism for the maintenance of meiotic arrest (Holt et al., 2013). In oocytes in the GV stage, a significant amount of cyclin B is already present. Therefore, the inactivation of CDK1 through phosphorylation is what maintains the oocyte in meiotic arrest (Holt et al., 2013). However, the cyclin B needs to be constantly degraded by the anaphase promoter complex/cyclossome (APC/C), in order to maintain blockage at prophase I. If the cyclin B accumulates during the GV stage, the increase in its concentration will activate the MPF leading to spontaneous resumption of meiosis (Reis et al., 2006). Meiosis resumption Re-initiation of meiosis depends upon several external factors. In mammals oocyte maturation is induced by the withdrawal of the inhibitory influence of GC in vivo the LH surge causes the breakdown of the gap junctions between the oocyte and the GC in the preovulatory follicle (Eppig, 1991). Similarly, the removal of immature oocytes from the follicular environment can interrupt the transfer of regulators and metabolic support crucial for the maintenance of meiotic arrest, resulting in resumption of oocyte maturation.

LH

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The re-initiation of meiosis is regulated by changes in the pattern of phosphorylation of several proteins by specific kinases. Of importance is the activity of MPF (Masui and Markert, 1971), which is the universal cell cycle regulator of mitosis and meiosis (Nurse, 1990). The activation of MPF induces condensation of chromosomes, breakdown of the nuclear envelop (GVBD) and preparation of the cytoplasm for the M phase during both mitotic and meiotic cycles (Murray, 1989; Murray and Kirschner, 1989; Motilik and Kubelba, 1990). In rodents and ruminants, the receptor for LH (LHR) is expressed mainly on cells of the theca and mural granulosa layers. A paracrine signal, as well as intercellular communication is crucial for the COC to respond to the LH surge (Peng et al., 1991). The effect of LH is to promote a decrease in the expression of NPPC receptors in CC and the consequent transfer of cGMP to the oocyte (Robinson et al., 2012). At the same time the gap junctions between the oocyte and CC are disrupted by factors from the EGF family such as the epiregulin, ampiregulin and beta-celulin (Norris et al., 2009; Vaccari et al., 2009). As a result of the drop in cGMP, there is an increase of PDE3 activity which promotes a rapid decline of cAMP levels in the oocyte and re-initiation of meiosis (Holt et al., 2013; Fig. 2). The decrease in cAMP levels results in a reduction of PKA activity and the Cdc25 is transferred to the nucleus (Oh et al., 2010). The accumulation of phosphatase Cdc25 in the nucleus promotes the activation of the MPF and the transportation of Wee1B to the cytoplasm. As meiosis is reinitiated, the Wee1B is inactivated and the Cdc25 is activated promoting an increase of CDK1 activity (Conti et al., 2012).

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inactive active cAMP cGMP Figure 2. Schematic representation of the events taking place during the resumption of oocyte meiosis. There is a reduction in the levels of cAMP, activation of MPF, germinal vesicle breakdown (GVBD) and oocyte maturation. CG= mural granulosa cells; CC= cumulus cells; Oo= oocyte; PDE= phosphodiesterase, EGFR= EGF receptor; GPR= receptor linked to the protein G; NPPC= natriuretic peptide, NPR2= receptor of natriuretic peptide; P= phosphorylation, Cx= conexin. Modified from Conti et al., 2012. 152

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Landim-Alvarenga and Maziero. Oocyte maturation.

Mitogen-Activated Protein Kinase (MAPKs) A group of proteins belonging to the serine/treonine family, the MAPKs, is involved in the progression of meiosis. These proteins are activated by extracellular signals and therefore are also known as ERK (extra cellular signal regulated kinase) with the variants ERK1 - p44 kDa and ERK2 - p42 kDa; Kubelka et al., 2000; Bilodeau-Goeseels, 2012). The MAPKs have several substrates similar to phosphorylation, including phospholipases, transcription factors and cytoskeleton proteins. The MAPK path is universally activated during meiotic maturation of vertebrate oocytes. In cattle, MAPK increases after 8 h of in vitro culture, continuing gradually until 12-14 h, and then remaining stable until maturation is completed (Kubelka et al., 2000; Quetglas et al., 2010). In bovine oocytes, the two main isoforms (ERK1/2) of MAPK are activated near the time of GVBD (Kubelka et al., 2000; Quetglas et al., 2010). This suggests that MAPK is not required for the initiation of meiosis, but is crucial for the post- GVBD events (Kubelka et al., 2000; Ponderato et al., 2001). The MAPK are found in the oocyte where they are activated by MOS kinase and in CC where they are activated by RAS/RAF. In both cell types the MAPK are activated by phosphorylation of tyrosine and treonine residues and by MEK, also named MAPKK (Mitogenactivated protein kinase kinase). Activation of MEK is also mediated by phosphorylation and the proteins MOS in the oocyte and RAS/RAF in the CC (Crocomo et al., 2013). In the mouse, LH induces the phosphorylation of ERK1/2 in pre-ovulatory follicles 30 min after stimulation and phosphorylation levels increase after 2 h (Panigone et al., 2008). The activation of the ERK1/2 occurs first in the mural GC and later in CC. The MAPK, when activated, promotes the MPF stabilization in oocytes through the inhibition of some negative regulators and activation of cdc25 phosphatase. In bovine oocytes, activation of MAPK occurs at the same time as or slightly before GVBD, with levels increasing gradually during oocyte maturation and remaining elevated until meiosis II. The activation and inactivation of MAPK is also related to variation in cAMP and PKA in the oocyte and CC. According to Sun et al. (2002), the activation of CC depends on paracrine factors secreted by the oocyte, showing the ability of this cell to control its own meiotic maturation. Nuclear maturation The process of nuclear maturation begins when meiosis resumes from the diplotene stage, signaled by chromosome condensation and GVBD. It corresponds to the reversal of the first blockage of meiosis from the GV stage until the second blockage at metaphase II

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(MII). After GVBD, the oocyte goes through metaphase I (MI), anaphase I (AI) and telophase I (TI), ending with the first meiotic division and then rapidly passes through MII of second meiotic division where a second meiotic arrest occurs (second meiotic blockage; Kubelka et al., 2000). By the end of the first meiotic division, homologues of oocyte chromosomes (2n) have separated into two nuclei with n chromosomes each (MI). The cytoplasm divides asymmetrically generating two cells, one keeping almost all the cytoplasm, the secondary oocyte and the other very small, the first polar body. Soon after, the second meiotic division begins and remains in this stage (MII) until the fertilization or parthenogenetic activation (Quetglas et al., 2010). The stage of nuclear maturation might be evaluated directly by the configuration of the chromosomes (Fig. 3) and classified in different stages (Landim-Alvarenga, 1999): • Germinal vesicle (GV): characterized by the presence of a spherical nucleus with intact envelope and filamentous chromatin; • Germinal vesicle breakdown (GVBD): characterized by condensed chromatin and absence of a visible nuclear membrane; • Metaphase I (MI): chromosomes arranged on the metaphase plate peripherally located in the ooplasm; • Metaphase II (MII): characterized by the presence of metaphase plate with chromosomes arranged in the periphery of the ooplasm and by the extrusion of the first polar body (PB) represented by a dense group of chromosomes. The integrity of the nuclear membrane including the GV is maintained by proteins called laminins. During GVBD, the CDK1 promotes disorganization of the nuclear envelop, phosphorylating the laminins (Adhikari and Liu, 2014). At same time as nuclear envelop disintegration, condensation of the chromosomes occurs and the metaphasic plate is organized. The protein degradation which occurs during the transition from metaphase to anaphase is controlled by the anaphase promoter complex (APC) which is responsible for the ubiquitination of several protein substrates (Sullivan and Morgan, 2007). The APC is a multi-subunit of the ubiquitin ligase E3, whose substrates are degraded by the proteosome 26S. The action of APC requires the linking of a protein coactivator, the CDC20 or FZR1 which confers specificity to the enzyme. The link of APC to CDC20 allows the anaphase of mitosis or meiosis to continue by degradation of cyclin B1 (Jones, 2011). In vertebrate oocytes, the metaphase’ spindle organizes to lead to an unequal division of cytoplasm, which results in the expulsion of first PB. Parallel to the expulsion of the PB, there is a decrease in MPF activity,

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which shortly increases again inducing the organization of the metaphase’ spindle for the second meiotic division, without formation of a nuclear envelop, chromosome de-condensation or DNA replication. High levels of MAPK and MPF are needed for the maintenance of oocytes in MII since fertilization or parthenogenetic activation causes an abrupt intra-oocyte drop of both kinases and the completion of meiosis (Hashimoto and Kishimoto, 1988; Choi et al., 1991; Naito and Toyoda, 1991; Jelinkova et al., 1994; Dedieu et al., 1996; Taieb et al., 1997; Wu et al., 1997; Oh et

al., 1998). When a spermatozoon enters the oocyte, the chromosomes separate and the organization of the nuclear envelop terminates meiosis with the extrusion of the second PB (2nd PB). After extrusion of the 2nd PB fusion of female and male pro-nuclei occurs, which is the beginning of the embryonic development. In order to ensure normal development of the embryo, nuclear changes during the oocyte maturation and fertilization are needed, which are coordinated by movements of the genetic material and organelles, and by biochemical changes in the cytoplasm (Van Blerkom, 1991).

A

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C

D

Figure 3. Meiotic stages of oocytes stained with Hoechst 33342 evaluated with an inverted microscope with ultra-violet light. The oocytes are classified as: A) Germinal vesicle (GV); B) Germinal vesicle breakdown (GVBD); C) Metaphase I (MI) and D) Metaphase II (MII). Amplification 400X. Cytoplasmic maturation The progression of meiosis that characterizes nuclear oocyte maturation, does not by itself guarantee further embryonic development; additional cytoplasm modifications or cytoplasm maturation is also necessary (Ferreira et al., 2009). Cytoplasmic maturation includes structural and molecular changes occurring in the oocyte from the GV stage to the end of MII. Evaluation of maturation might be done indirectly through the ability of the mature oocyte to cleave and develop into a blastocyst after fertilization or parthenogenetic activation. The ultrastructural changes in the cytoplasm include migration of several of the organelles. The mitochondria and Golgi complex, which are located in the periphery of the oocyte in immature oocytes, move to a perinuclear distribution. The cortical granules, originating from the Golgi complex, and originally situated in the center of the oocyte migrate to the 154

periphery and become anchored membrane (Cran and Moor, 1990).

to

the

oocyte

Redistribution of organelles in the cytoplasm The location of the organelles in the cytoplasm depends on the cell’s needs at each stage of development and their relocation is made possible by the action of cytoskeleton microfilaments and microtubules. The resumption of meiosis is accompanied by extensive reorganization of the cytoplasmic microtubules in the oocyte. During interphase, long and relatively stable microtubules are distributed in the cytoplasm, while during metaphase, the microtubule organizer centers (MTOCs) are phosphorylated and the activity of microtubule organization increases (Fan and Sun, 2004). Thus, mitochondria, endoplasmic reticulum, Golgi complex and cortical granules assume different positions from those observed at GV stage (Ferreira et al., 2009). Anim. Reprod., v.11, n.3, p.150-158, Jul./Sept. 2014

Landim-Alvarenga and Maziero. Oocyte maturation.

Mitochondria The activation of metabolic pathways through protein synthesis and phosphorylation is indispensable for cytoplasmic maturation. The mitochondria have a very important role in this process since they are a key component of energy metabolism (Krisher and Bavister, 1998; Meirelles et al., 2004). In this sense, the dislocation of the mitochondria to areas of high energy consumption is crucial for oocytes and embryos in critical periods of the cellular cycle. During maturation, mitochondria synthesize ATP needed for the production of proteins used during late embryo development (Meirelles et al., 2004). A structural analysis of bovine oocytes submitted to in vitro maturation (IVM) show that, after 12 to 18 h in culture, the mitochondria change from a position more peripheral to a dispersed location throughout the cytoplasm (Hyttel et al., 1986). This behavior is similar to what occurs in vivo, which means that the distribution is more peripheral before the LH peak, beginning to diffuse during the final phases of nuclear maturation and then dispersing after the polar body extrusion, approximately 19 h post-LH peak (Kruip et al., 1983; Hyttel et al., 1997). Studies of bovine and murine oocytes indicate that the reorganization of mitochondria in cytoplasm post-IVM is correlated to the ATP levels in the embryos. Therefore, embryos with less ATP in the cytoplasm develop more slowly and have lower number of cells (Liu et al., 2000). Before the embryonic genome is activated (after 72 h of culture) the mitochondria has an intermediate level of activity, which might be explained by the adaptive protection against the reactive types of oxygen (ROs) This protection occurs as a result of catalyzer molecules such as glutathione and peroxidases, which are produced during oocyte maturation and early embryonic development (Krisher and Bavister, 1998). Besides the activities described above, the mitochondria regulate the process of cellular apoptosis, acting as reservoirs of activator proteins of programmed cell death (PCD) process, as for example cytocrome c. Permeabilization of the mitochondrial membrane allows the liberation of cytocrome and consequent activation of the apoptosis cascade resulting in cell death (Van Blerkom, 2004). Endoplasmic reticulum The endoplasmic reticulum (ER) membranes are physiologically active, containing specialized domains interacting with the cytoskeleton in the accomplishment of different functions. Among the known functions of the ER are the folding and degradation of proteins, lipid metabolism, nucleus compartmentalization, establishment of calcium (Ca2) Anim. Reprod., v.11, n.3, p.150-158, Jul./Sept. 2014

gradients, and its own synthesis (Ferreira et al., 2009). Throughout calcium storage, the system plays an important role in intracellular signaling. The mechanisms and paths of calcium ion (Ca2) mobilization point to its importance in several cellular events. The paths of Ca2 signaling are dependent on differences in its extra and intra cellular contents, which are responsible for concentration gradients between both compartments. The established gradient is regulated by the ooplasm membrane. In rodent and human oocytes the content of Ca2 in the cytoplasmic reticulum is mediated by proteins present in the reticulum canals; the receptors for inositol 1,4,5 triphosphate (IP3R) and ryanodine are both located in the ER membrane and are responsible for the control of Ca2 movement into the cytosol. The Ca2 liberation via IP3 and its receptor IP3R is crucial for oocyte activation during fertilization (Kline and Kline, 1994). Biochemical and structural modifications of the ER during maturation are essential for the satisfactory functioning of intracellular calcium regulation. Examination of mouse oocytes in the GV stage shows that the ER are uniformly distributed in the ooplasm. During the progression of development until the MII stage, the ER are found in the cortical region accumulating in small stacks of 1-2 um over all cytoplasm (Stricker, 2006). The system sensibility to the needs of Ca2 liberation increases after maturation. During fertilization, the entrance of the spermatozoon into the oocyte provokes the exit of Ca2 from the ER which is followed by the beginning of embryonic development (Ferreira et al., 2009). Golgi Complex (GC) The dynamics of Golgi membranes during maturation and fertilization of mammalian oocytes requires additional research. In the GV oocyte, the Golgi complex appears in the periphery of the ooplasm surrounded by small vesicles. Two kinds of vesicles are observed: coated vesicles resembling pinocytosis granules are seen next to the cis face of the Golgi complex while, next to the trans face smooth vesicles with irregular size and electron density are observed (Landim-Alvarenga and Alvarenga, 2006). Electron dense membranous granules appeared in association with the Golgi complex or distributed through the cytoplasm which were classified as cortical granules. On the other hand, in matured oocytes, Golgi complexes were still present but less developed (Landim-Alvarenga and Alvarenga, 2006). Cortical granules The cortical granules (CG) originate in the Golgi complex. The exocytosis of the CGs involves cytoskeleton filaments and homologue proteins. In GV 155

Landim-Alvarenga and Maziero. Oocyte maturation.

oocytes, the cortical granules are in clusters through the cytoplasm. By the end of the maturation period, when MII is attained, the granules are near the inner surface of oocyte cell membrane. This pattern is a strategic distribution in preparation for arrival of the spermatozoon and oocyte activation (Hosoe and Shioya, 1997). The cortical granules are organelles exclusive of oocytes and their composition consists of a variable population of proteins, structural molecules, enzymes and glycosaminoglycans. The exocytosis of the cortical granules (cortical reaction) is one of the mechanisms most frequently used by the oocytes to avoid polyspermy. If the oocyte is fertilized by more than one spermatozoon, the resulting zygote will undergo abnormal cleavage, becoming nonviable and will degenerate as soon as the mitotic divisions begin (Hosoe and Shioya, 1997). Molecular maturation Molecular maturation consists of several stages such as transcription, storage and processing of the mRNA that will be used in ribosome synthesis of the proteins that will directly influence the subsequent cellular events, such as fertilization, pro-nucleus formation, and the beginning of the embryogenesis (Crocomo et al., 2013). Transcription and mRNA storage occur during the folliculogenesis, while the nucleus is quiescent, and ends when meiosis is reinitiated, soon after the chromosomes condense and became inactive. However, the capacity of mRNA translation and protein synthesis is maintained throughout oocyte development and further embryogenesis (Sirard, 2001). Most of the ooplasm mRNA is stable, but inactive due to its short poly-A tail. Under the action of signals generated during maturation, fertilization and the beginning of embryo development, there is polyadenylation, which is the addition of adenines to the 3’end under the action of the poly-a polymerase. Polyadenylation promotes the release of repressor molecules coupled to the 5’segment, allowing for the beginning of translation (Gottardi and Mingoti, 2009). The transportation of mRNA to the cytoplasm occurs as a result of a characteristic shortening of the poly-A tail, which upon reaching this compartment becomes shorter and heterogeneous (Tomek et al., 2002). The mRNA molecules are not translated when they have short poly-A tails. Therefore, deletion of that sequence is the initial step in their degradation process (Tomek et al., 2002). The cytoplasm elongation of the poly-A tail means the activation of translation, which is, the addition of adenine to mRNA in the oocyte cytoplasm during maturation, leading to protein synthesis, and deadenylation for degradation of those mRNA (Ferreira et al., 2009). Therefore, the efficiency of storage and 156

reactivation of mRNAs, is regulated by polyadenylation and determines oocyte competence to support later developmental stages. The pronounced increase of kinase activity initiates a complex and specific cascade of protein phospho-dephosphorylation (Gottardi and Mingoti, 2009). Conclusion The information reviewed above shows that the process of maintenance of meiotic arrest involves a complex system of cellular signals that is modified by the pre-ovulatory stimulus of LH, resulting in reinitiation of meiosis and oocyte maturation. Most of the events reported here are intracellular, while others involve paracrine controls depending on intimate relations between follicular cells and the oocyte. All events are necessary to the production of a functional gamete with the capacity to develop to a healthy embryo after fertilization. References Adhikari D, Liu K. 2014. The regulation of maturation promoting factor during prophase I arrest and meiotic entry in mammalian oocytes. Mol Cell Endocrinol, 382:480-487. Bilodeau-Goeseels S. 2012. Bovine oocyte meiotic inhibition before in vitro maturation and its value to in vitro embryo production: does it improve developmental competence? Reprod Domest Anim, 47:687-693. Bornslaeger EA, Mattei P, Schultz RM. 1986. Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev Biol, 114:453-462. Choi T, Aoki F, Mori M, Yamashita M, Nagahama Y, Kohmoto K. 1991. Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development, 113:789795. Conti M, Andersen CB, Richard F, Mehats C, Chun SY, Horner K, Jin C, Tsafriri A. 2002. Role of cyclic nucleotide signaling in oocyte maturation. Mol Cell Endocrinol, 187:153-159. Conti M, Hsieh M, Zamah AM, Oh JS. 2012. Novel signaling mechanisms in the ovary during oocyte maturation and ovulation. Mol Cell Endocrinol, 356:6573. Cran DG, Moor RM. 1990. Programming the oocyte for fertilization. In: Bavister BD, Cummins J, Roldan ERS (Ed.). Fertilization in Mammals. Norwell, MA: Serono Symposia. pp. 35-50. Crocomo LF, Marques Filho WC, Sudano MJ, Paschoal DM, Landim-Alvarenga FC, Bicudo SD. 2013. Effect of roscovitine and cycloheximide on ultrastructure of sheep oocytes. Small Rumin Res, 109:156-162. Dedieu T, Gall L, Crozet N, Sevellec C, Ruffini S. Anim. Reprod., v.11, n.3, p.150-158, Jul./Sept. 2014

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Reprod Dev, 37:210-215. Jones KT. 2011. Anaphase-promoting complex control in female mouse meiosis. Results Probl Cell Differ, 53:343-363. Kline JT, Kline D. 1994. Regulation of intracellular calcium in the mouse egg: evidence from inositol trisphosphate-induced calcium release, but not calciuminduced calcium release. Biol Reprod, 50193-203. Krisher RL, Bavister BD. 1998. Responses of oocytes and embryos to the culture environment. Theriogenology, 59:103-114. Kruip TAM, Cran DG, Van Beneden TH, Dieleman SJ. 1983. Structural changes in bovine oocytes during final maturation in vivo. Gamete Res, 8:29-47. Kubelka M, Motlík J, Schultz RM, Pavlok A. 2000. Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes, without influencing chromossome condensation activity. Biol Reprod, 62:292-302. Landim-Alvarenga FC. 1999. Produção in vitro de embriões equinos: avanços e limitações. Arq Fac Vet UFRGS, 27:54-89. Landim-Alvarenga FC, Alvarenga MA. 2006. Structural aspects of equine oocytes matured in vivo and in vitro. Braz J Morphol Sci, 23:513-524. Lew DJ, Kornbluth S. 1996. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol, 8:795-804. Liu L, Trimarchi JR, Keefe DL. 2000. Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes. Biol Reprod, 62:1745-1753. Maller JL. 1980. Regulation of oocyte maturation. Curr Top Cell Regul, 16:271-311. Masui Y, Markert CL. 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zool, 177:129-146. Meirelles FV, Caetano AR, Watanabe YF, Ripamonte P, Carambula SF, Merighe GK. 2004. Genome activation and developmental block in bovine embryos. Anim Reprod Sci, 82/83:3-20. Motlík A, Kubelka M. 1990. Cell cycle aspects of growth and maturation of mammalian oocytes. Mol Reprod Dev, 27:366-375. Murray AW. 1989. The cell cycle as a cdc2 cycle. Nature, 342:14-15. Murray AW, Kirschner MW. 1989. Domineos and clocks: the union of two views of the cell cycle. Science, 246:614-621. Naito K, Toyoda Y. 1991. Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J. Reprod. Fertil., 93:467-473. Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA,Wang H, Ke H, Nikolaev VO, Jaffe LA. 2009. Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development, 136:18691878. Nurse P. 1990. Universal control mechanism regulating onset of M-phase. Nature, 344:503-508. 157

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Oh B, Hampl A, Eppig JJ, Solter D, Knowles BB. 1998. Spin, a substrate in the MAP kinase pathway in mouse oocytes. Mol Reprod Dev, 50:240-249. Oh JS, Han SJ, Conti M. 2010. Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption. J Cell Biol, 188:199-207. Panigone S, Hsieh M, Fu M, Persani L, Conti M. 2008. Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway. Mol Endocrinol, 22:924-936. Paynton BV, Bachvarova R. 1990. Changes in maternal RNAs during oocyte maturation. In: Bavister, BD, Cummins J, Roldan ERS. (Ed.). Fertilization in Mammals. Norwell, MA: Serono Symposia. pp. 25-34. Peng XR, Hsueh AJ, LaPolt PS, Bjersing L, Ny T. 1991. Localization of luteinizing hormone receptor messenger ribonucleic acid expression in ovarian cell types during follicle development and ovulation. Endocrinology 129:3200-3207. Pirino G, Wescott MP, Donovan PJ. 2009. Protein kinase A regulates resumption of meiosis by phosphorylation of Cdc25B in mammalian oocytes. Cell Cycle 8:665-670. Ponderato N, Lagutina I, Crotti G, Turini P, Galli C, Lazzari G. 2001. Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity. Mol Reprod Dev, 60:579-585. Quetglas MD, Adona PR, de Bem THC, Pires PRL, Leal CLV. 2010. Effect of cyclin-dependent Kinase (CDK) inhibition on expression, localization and activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) in bovine oocytes. Reprod Domest Anim, 45:1074-1081. Reis A, Chang HY, Levasseur M, Jones KT. 2006. APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division. Nat Cell Biol, 8:539-540. Robinson JW, Zhang M, Shuhaibar LC, Norris RP, Geerts A, Wunder F, Eppig JJ, Potter LR, Jaffe LA. 2012. Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes. Dev Biol, 366:308-316. Sathananthan AH. 1994. Ultrastructural changes during meiotic maturation in mammalian oocytes: unique aspects of the human oocyte. Microsc Res Tech, 27:145-164, Schultz RM, Lamarca MJ, Wassarman PM. 1978. Absolute rates of protein synthesis during meiotic maturation of mammalian oocytes in vitro. Proc Natl

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Anim. Reprod., v.11, n.3, p.159-167, Jul./Sept. 2014

Effect of uterine environment on embryo production and fertility in cows A.H. Souza1,5, C.D. Narciso2, E.O.S. Batista1,3, P.D. Carvalho4, M.C. Wiltbank4 1

University of California, Cooperative Extension, Tulare, CA, USA. Veterinarian practitioner, Sequoia Veterinary Services, Tulare, CA, USA. 3 Department of Animal Reproduction, FMVZ, University of São Paulo, SP, Brazil. 4 Department of Dairy Science, University of Wisconsin-Madison, WI, USA. 2

Abstract Oocyte fertilization rates in bovines following artificial insemination or natural mating are generally good (~90%). Curiously, only about one third of these pregnancies remain until 30 days post-AI in dairy cows. Thus, most pregnancies are lost between fertilization and early embryonic growth. Although classical pathways describing that lower progesterone post-AI is the main culprit to these early embryonic losses, a number of environmental factors such as heat-stress as well as novel concepts in bovine physiology including the effects of excessive negative energy balanced (NEB) and the insulin-resistant state experienced by high producing cows during the postpartum period can help explain the poor reproductive performance, generally observed in dairy herds world-wide. Thus, expanding the scientific knowledge in these critical areas in bovine fertility related to the evident impact of NEB and/or altered circulating and uterine metabolites in the postpartum period on oocyte quality, gamete transport, uterine environment, and early embryonic growth are of major importance to improve reproductive efficiency in modern high producing dairy cows. Keywords: dairy cow, fertility, oocyte, uterus. Introduction Early embryo development and fertility of modern lactating dairy cows is far less than ideal, with only about 50% of the embryos been reported to be viable by 7 days after ovulation as compared to ~80% in non-lactating cows (Sartori et al., 2009). However, in the last couple of decades, we have gained important insights in understanding some of the complex interactions between the cow’s environment, management, nutrition, level of milk production and possible breeding strategies that may improve overall fertility. For example, a growing body of evidences indicates that low circulating progesterone (P4) after ovulation in dairy cows seems to be related to conceptus growth and most likely mediated through changes in the endometrium rather than directly in the embryo (Clemente et al., 2009). However, several other blood parameters and management issues such as heat-stress _________________________________________ 5 Corresponding author: [email protected] Phone: +1(559)684-3313; Fax+1(559)685-3319 Received: July 1, 2014 Accepted: July 18, 2014

may alter uterine environment, oocyte quality and embryo development. Thus, the aim of this manuscript is to review some factors associated with poor uterine health, embryo quality/development and fertility of dairy cows. It is important to highlight that multiple nutritional factors have been also associated with inadequate uterine environment, oocyte quality, and/or cow-immunity and health including diets that produce high levels of blood urea nitrogen (BUN), nutritional contaminants such as gossypol or mycotoxins, varying effects of different fat-sources as reviewed in Bisinotto et al. (2012). However, reviewing possible nutritional components that may influence fertility of dairy cows is out of the scope of the current manuscript. Emphasis will be given though to the detrimental impact of uterine infections or of excessive body weight losses that may occur in the postpartum period, with major direct effects in follicular dynamics, uterine tract physiology, embryo quality and ultimately conception results of dairy cows. A model to study the impact of uterine environment of lactating cows Trying to isolate areas in the whole body physiology and/or specific parts that might be responsible for poor fertility within the reproductive tract in bovines is a challenging task. For example, several research groups have described poor embryo quality and conception rate results in lactating cows, but it is unclear whether early embryonic growth up to day 7 post ovulation are reduced due to inadequate uterine environment and/or due to (i.e.) overexposure of oocytes to longer periods of high LH pulsatility - both hypothesis seem fairly plausible and could potentially be related to high milk production levels. Interestingly, it appears that embryo growth following day 7 post ovulation is compatible with high volumes of daily milk production since a growing body of scientific literature (Demetrio et al., 2007) supports the concept that the use of embryo transfer into lactating cows on day 7 seem to improve fertility in relation to regular AI. Further evidence to that is the fact that attempts to increase P4 levels after day 7 generally yield marginal to no results (Nascimento et al., 2013). These elements argue for at least nearly normal uterine environment to support pregnancy to term from day 7 to calving, and suggest

Souza et al. Uterine environment and fertility in cows.

that most issues with fertility of lactating cows is related to poor oocyte quality and/or inadequate uterine environment to support early embryo growth before day 7. Although recent findings argue for significant importance of some blood parameters (i.e. glucose) for embryo development after day 7 (Green et al., 2012). Despite of that, later findings indicate that oocyte quality can be improved in lactating cows through greater pre-ovulatory circulating P4 concentrations (Wiltbank et al., 2012), but little is actually known about the capacity of the uterus of lactating cows to cope with adequate fertilization and early embryo survival until day 7 after ovulation. Although scarce, later publications were able to shed light on the events within the uterine tract from day 2 to day 7 after ovulation by utilizing laparoscopic transfer of IVF embryos into the oviduct (Rizos et al., 2010; Maillo et al., 2012). Both reports described drastic reductions in proportion of embryos that remained viable from day 2 to day 7 of the estrous cycle when these embryos were transferred into lactating cows as compared to nulliparous heifers (Rizos et al., 2010) or postpartum cows that were dried-off at calving (Maillo et al., 2012). The authors hypothesize that a combination of factors and complex associations may result in less than ideal endometrial environment in the lactating cow. Such factors are high milk production inducing low circulating P4 due to greater steroid metabolism, negative energy balance that lactating cows undergo at beginning of lactation, with remarkable increase in circulating non-esterified fatty acids (NEFA) as well as lower blood calcium, glucose and IGF-1. Lower circulating P4 before and/or after ovulation has been extensively studied and associated with embryonic growth and conception results (Wiltbank et al., 2012); however, other blood parameters such as calcium and glucose concentrations have been largely overlooked and only recently studied more in depth to unravel their importance on dairy cow fertility. For example, low circulating calcium concentrations ( 0.10) to incidence of subclinical endometritis. These results corroborate with findings from a recent study (Scully et al., 2013), in which they found no significant differences in uterine diameter and fluid volume by ~50 DIM in cows that were lactating or dried-off just after calving time to try to isolate effects of lactation on uterine environment. Thus, milk production per se does not seem to be the major factor associated with the capacity of cows to undergo uterine involution postpartum. Surprisingly though, based on the results from Carvalho et al. (2013), it appears that greater proportions of PMN in uterine lumen have a direct effect in a number of embryo production parameters, as shown in Table 1 and Fig. 1. Thus, results from Carvalho et al. (2013) and Scully et al. (2013) provide compelling evidence that milk production has little effect on uterine health, but poor uterine environment (greater proportion of PMNs) can greatly impair oocyte fertilization capacity. Obviously, the underlying physiology related to poor conception results in lactating cows lie in multiple factors including uterine infections that may disrupt normal postpartum follicular growth and delayed resumption of ovarian activity (anovulation), and/or direct effects in the uterine environment. It appears that the whole physiology of negative energy balance alongside with alterations in insulin signaling and IGF system, as well as deviations in circulating calcium in the early postpartum cow can have a major Anim. Reprod., v.11, n.3, p.159-167, Jul./Sept. 2014

Souza et al. Uterine environment and fertility in cows.

impact in the process of normal immunity and uterine involution (Wathes et al., 2011; Martinez et al., 2012). For example, Wathes et al. (2011) randomized postpartum lactating cows to undergo mild or severe negative energy balance. Interestingly, these researchers described great upregulation patterns in several genes linked to metalloproteinase activity - an intrinsic part of uterine remodeling postpartum; indicating that uterine involution of cows under greater

NEB seem to be altered and these deviations from normal physiology are likely caused by alterations in the IGF1/insulin signaling pathways in the endometrium of cows undergoing NEB. Thus, postpartum is characterized by a period of uterine tissue remodeling, and efforts to avoid excessive NEB and body loss experienced by cows during the first couple of weeks postpartum should improve uterine health and ultimately fertility.

Table 1. The effect of uterine polymorphonuclear cells (PMN) on ova/embryo recovery, fertilization, and transferable/freezable embryo numbers. PMN PMN PMN Endpoint 5% P-value (n = 40) (n = 13) (n = 12) CL number

17.7 ± 1.4

Total ova/embryos recovered

7.8 ± 1.1

% Recovery

ab

41.5 ± 4.3

Fertilized structures

82.3 ± 3.4

Transferable embryos

17.2 ± 1.7

0.84

9.2 ± 2.4

4.7 ± 1.1

0.10

55.5 ± 8.5

5.9 ± 7.7a

% Fertilized structures

15.8 ± 2.3

4.6 ± 0.7a

a

28.4 ± 6.3

b

0.04

7.4 ± 1.9a

2.3 ± 0.7b

150 pg/mL; n = 9). Administration of PG significantly increased the number of: small follicles (2-3 mm) and total follicles (2-8 mm) on day 2 of the cycle in all heifers. PG improved in vitro embryonic development rate (total number of embryos/number of fertilized oocytes) in all heifers compared to the control. Taken together, these findings open-up the possibility of improving fertility and embryo quality by using diets or dietary supplements which induce a programmed sequence in circulating insulin concentrations.

Insulin related feeding strategies References Exogenous insulin administration increased the recruitment of follicles in response to gonadotropin in gilts (Cox et al., 1987) and also rescues follicles from 196

Adamiak SJ, Mackie K, Watt RG, Webb R, Sinclair KD. 2005. Impact of nutrition on oocyte quality: Anim. Reprod., v.11, n.3, p.195-198, Jul./Sept. 2014

Ponsart et al. Circulating insulin and embryo development.

cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biol Reprod, 73:918-926. Armstrong DG, McEvoy TG, Baxter G, Robinson JJ, Hogg CO, Woad KJ. 2001. Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insulin-like growth factor system. Biol Reprod, 64:1624-1632. Bender RW, Hackbart KS, Dresch AR, Carvalho PD, Vieira LM, Crump PM, Guenther JN, Fricke PM, Shaver RD, Combs DK, Wiltbank MC. 2014. Effects of acute feed restriction combined with targeted use of increasing luteinizing hormone content of follicle-stimulating hormone preparations on ovarian superstimulation, fertilization, and embryo quality in lactating dairy cows. J Dairy Sci, 97:764-778. Boland MP, Lonergan P, O’Callaghan D. 2001. Effect of nutrition on endocrine parameters, ovarian physiology and oocyte and embryo development. Theriogenology, 55:1323-1340. Cox NM, Stuart MJ, Althen TG, Bennett WA, Miller HW. 1987. Enhancement of ovulation rate in gilts by increasing dietary energy and administering insulin during follicular growth. J Anim Sci, 64:507-516. Freret S, Grimard B, Ponter AA, Joly C, Ponsart C, Humblot P. 2006. Reduction of body weight gain enhances in vitro production in overfed superovulated dairy heifers. Reproduction, 131:783-794. Gamarra G, Ponsart C, Lacaze S, Le Guienne B, Deloche M-C, Monniaux D, Ponter A. 2014a. Short term dietary propylene glycol supplementation affects circulating metabolic hormones, progesterone concentrations and follicular growth in dairy heifers. Livest Sci, 162:240-251. Gamarra G, Ponsart C, Lacaze S, Le Guienne B, Humblot P, Deloche M-C, Monniaux D, Ponter A. 2014b. Effect of dietary propylene glycol on ovarian follicle growth, the superovulatory response and in vitro embryo production after ovum pick-up in growthrestricted heifers differing in their plasma AMH profiles. Reprod Fertil Dev. (accepted for publication). Garnsworthy PC, Sinclair KD, Webb R. 2008. Integration of physiological mechanisms that influence fertility in dairy cows. Animal, 2:1144-1152. Garnsworthy PC, Fouladi-Nashta AA, Mann GE, Sinclair KD, Webb R. 2009. Effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows. Reproduction, 137:759-768. Gong JG, McBride D, Bramley TA, Webb R. 1993. Effects of recombinant bovine somatotrophin, insulinlike growth factor-I and insulin on the proliferation of bovine granulosa cells in vitro. J Endocrinol, 139:67-75. Gong JG, Armstrong DG, Baxter G, Hogg CO, Garnsworthy PC, Webb R. 2002a. The effect of increased dietary intake on superovulatory response to FSH in heifers. Theriogenology, 57:1591-1602. Gong JG, Lee WJ, Garnsworthy PC, Webb R. Anim. Reprod., v.11, n.3, p.195-198, Jul./Sept. 2014

2002b. The effect of dietary induced increases in circulating insulin concentrations during the early post partum period on reproductive function in dairy cows. Reproduction, 123:419-427. Gutierrez CG, Oldham J, Bramley TA, Gong JG, Campbell BK, Webb R. 1997. The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers. J Anim Sci, 75:1876-1884. Landau S, Braw-Tal R, Kaim M, BorA,Bruckental I. 2000. Preovulatory follicular status and diet affect the insulin and glucose content of follicles in high-yielding dairy cows. Anim Reprod Sci, 64:181-197. Lozano JM, Lonergan P, Boland MP, O’Callaghan D. 2003. Influence of nutrition on the effectiveness of superovulation programmes in ewes: effect on oocyte quality and post - fertilization development. Reproduction, 125:543-553. Lucy MP. 2000. Regulation of ovarian follicular growth by somatotropin and insulin growth factors in cattle. J Dairy Sci, 83:1635-1647. Majerus V, De Roover R, Etienne D, Kaidi S, Massip A, Dessy F, Donnay I. 1999. Embryo production by ovum pick up in unstimulated calves before and after puberty. Theriogenology, 52:1169-1179. Mantovani R, Enright WJ, Keane MG, Roche JF, Bolan MP. 1993. Effect of nutrition and dose of follicle stimulating hormone (FSH) on superovulatory response in beef heifers. In: Proceedings of the 9th AETE Meeting, 1993, Lyon, France. Lyon: AETE. abstr. 234. Maplesden DC. 1954. Propylene glycol in the treatment of ketosis. Can J Comp Med Vet Sci, 1:287293. Matamoros IA, Cox NM, Moore AB. 1991. Effects of exogenous insulin and body condition on metabolic hormones and gonadotropin-induced follicular development in prepuberal gilts. J Anim Sci, 69:20812091. Matoba S, Bender K, Fahey AG, Mamo S, Brennan L, Lonergan P, Fair T. 2014. Predictive value of bovine follicular components as markers of oocyte developmental potential. Reprod Fertil Dev, 26:337345. McGuire MA, Dwyer DA, Harrell RJ, Bauman DE. 1995. Insulin regulates circulating insulin-like growth factors and some of their binding proteins in lactating cows. Am J Physiol, 269: E723-E730. Nielsen NI, Ingvartsen KL. 2004. Propylene glycol for dairy cows a review of the metabolism of propylene glycol and its effects on physiological parameters, feed intake, milk production and risk of ketosis. Anim Feed Sci Technol, 115:191-213. O’Callaghan D, Yaakub H, Hyttel P, Spicer LJ, Boland MP. 2000. Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone concentrations in ewes. J Reprod Fertil, 118:303-313. Papadopoulos S, Lonergan P, Gath V, Quinn KM, Evans AC, O’Callaghan D, Bolan MP. 2001. Effect of 197

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diet quantity and urea supplementation on oocyte and embryo quality in sheep. Theriogenology, 55:1059-1069. Santos J, Cerri R, Sartori R. 2008. Nutritional management of the donor cow. Theriogenology, 69:88-97. Scaramuzzi RJ, Campbell BK, Downing JA, Kendall NR, Khalid M, Muñoz-Gutiérrez M,Somchit A. 2006. A review of the effects of supplementary nutrition in the ewe on the concentrations of reproductive and metabolic hormones and the mechanisms that regulate folliculogenesis and ovulation rate. Reprod Nutr Dev, 46:339-354. Seneda M Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery. Anim Reprod Sci, 67:37-43. Wathes DC, Fenwick MA, Liewellyn S, Cheng Z, Fitzpatrick R, McCarthy SD, Morris DG, Patton J, Murphy JJ. 2008. Influence of energy balance on gene

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Different ways to evaluate bovine sexed sperm in vitro J.O. Carvalho1,3, R. Sartori1, M.A.N. Dode2 1

Department of Animal Science, ESALQ, University of São Paulo, Piracicaba, SP, Brazil. Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil.

2

Abstract Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry. Keywords: flow cytometry, in vitro assessment, sexing process, sperm. Introduction Sperm sexing has the potential to influence the birth rate of the desired gender, allowing greater production efficiency and flexibility in herd management. Moreover, the possibility to choose the gender of offspring, even before embryo production or pregnancy, according to the needs of the livestock and/or market demands, results in greater economic gain (Wheeler et al., 2006). Although several methods have been developed for sperm sex determination (Koo et al., 1973; Kaneco et al., 1984; Johnson et al., 1987), the only method effective for routine use is the fluorescence-activated cell sorting using flow cytometry. This method is based on differences on DNA content of X and Y chromosome-bearing sperm cells (Garner et al., 1983), and usually has 90% of accuracy (Seidel and Garner, 2002). Bovine sperm prepared by this method (for method description see Seidel and Garner, 2002) is available commercially in Brazil since 2006, and since _________________________________________ 3 Corresponding author: [email protected] Phone: +55(61)9609-1270 Received: May28, 2014 Accepted: July 11, 2014

then its use has increased markedly. Part of this growth can be attributed to its wide use in the in vitro embryo production (IVF), which is one of the most advantageous combinations of reproductive biotechnologies. In other words, the sexed sperm is gaining more and more space and, like other reproductive biotechnologies, has become almost an indispensable procedure for those who want to keep high production and economically performance, especially in the dairy industry. This makes a real economic sense because recipient resources would not be wasted to produce calves of the unwanted gender (Butler et al., 2014). Although sexed sperm is currently used, the high cost and the reduced pregnancy rates compared to conventional sperm, have been limiting its application in cattle breeding. This suggests that sexing process may induces sperm damages, which can be due to exposure to the laser, to the high velocity inside the collecting tube, to electric charges, and to room temperature before being processed (Garner, 2006; Wheeler et al., 2006). Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, an evaluation of the possible structural and functional damages caused by the sexing process is needed. Undoubtedly, the best way to assess the quality of sperm sample is through the pregnancy rate and/or birth after artificial insemination (AI). However, the high cost and time required to obtain the results, make those types of analysis almost unfeasible. Therefore, other techniques for in vitro evaluation of sperm have been developed in order to better predict fertility of those cells and to estimate most accurately quality of a sperm sample (Amann and Hammerstedt, 1993). Therefore, this review aims to present several analyses made in sexed sperm that can estimate its structural and functional viability. Effect of sexing process on sperm structure and function Assessment of motility According to Malmgren (1997), motility is an important factor to be considered in the analysis of sperm viability. Among the characteristics affected by the sexing process, decreased motility has been reported

Carvalho et al. In vitro evaluations of sexed sperm.

by several authors (Hollinshead et al., 2004; Blondin et al., 2009; Carvalho et al., 2009, 2010). Moreover, Carvalho et al. (2009) found lower straight-line velocity, beat-cross frequency and linearity assessed by computer-assisted semen analysis (CASA), in sexed than non sexed sperm. This change in the sexed sperm motility, could have been caused by exposure to Hoechst 33342 stain, the laser light, or exposure in the droplets to electric charges (Watkins et al., 1996). According to Smith (1993), the effect of exposure to dye and then, the laser, may reduce mitochondrial activity, causing a decrease in the production of ATP. According to Alomar et al. (2006), motility is one of the most important sperm characteristics for the maintenance of fertility, being its evaluation, either subjective or by computerized analysis, essential in any sperm analysis. Assessment of DNA, plasma membrane and acrosome integrity Besides motility, several other sperm features can be evaluated after sperm sexing procedure, such as changes in DNA. However, studies have shown that sexing does not affect sperm DNA integrity (Blondin et al., 2009; Carvalho et al., 2010; Gosálvez et al., 2011). It is well known that chromatin stability is related to the proportion of protamine: histone present in sperm chromatin which varies from 1% in the mouse (Balhorn et al., 1977) to 15% in the human (Gatewood et al., 1990) and over 50% in some marsupial species (Soon et al., 1977). Then, the high proportion of protamine: histone present in DNA of bovine sperm can be responsible by the high stability of the chromatin which protects the DNA against the possible damages of the sexing procedure. Additionally, it is important to indicate that the sexing process has improved in recent years (Sharpe and Evans, 2009) due to modifications on the process, such as decrease of sexing pressure, and the use of dye to exclude dead sperm, among others. Those changes made the process more efficient and less harmful to the sperm. This is supported by recent studies that reported similar structural and/or functional quality in sorted and non sorted bull sperm (Blondin et al., 2009; Peippo et al., 2009; Carvalho et al., 2010). Another physical characteristic that may be affected by sexing process is plasma membrane integrity. Results from different studies have shown that the sexing procedure increases the percentage of sperm with plasma membrane damaged (Blondin et al., 2009; Carvalho et al., 2010; Villamil et al., 2012; Spinaci et al., 2013). These effects may be due to mechanical stress (Garner, 2006), since it has been shown that a decrease in pressure during cell sorting increases the percentage of sperm with intact membrane, increasing fertilization (Suh et al., 2005) and pregnancy rates (Schenk et al., 2009). However, despite the fact that lower pressure minimize damage to sperm, it may 200

compromise the efficiency of the sexing process (Garner, 2006). Besides plasma membrane, the acrosome can also be affected by the sexing process, which can substantially impair the ability of sperm cells to fertilize the oocyte since an acrosome intact is necessary to bind to the zona pellucida and fertilize the oocyte. Using different techniques, such as fluorescent probe used to assess acrosome integrity by fluorescent microscope or flow citometry, studies have shown a higher (Mocé et al., 2006; Carvalho et al., 2010) or similar (Klinc and Rath, 2007; Blondin et al., 2009) percentage of cells with acrosome reacted in sexed sperm than in non sexed. This variation can be due to the different techniques used to evaluate the acrosome integrity (Brito et al., 2003). Moreover, the variation found in those results can also be attributed to individual sensibility of each bull to the sexing process. There are large numbers of possibilities to evaluate structural characteristics of sperm cell using different staining methods and different types of equipments. A simple assessment can be performed with vital dyes such as Eosin/nigrosin, Trypan blue and Giemsa, which can be assessed by light microscopy or phase contrast. However, this type of procedure tends to underestimate the percentage of damaged sperm. Therefore, to have a more accurate detection of the different structural characteristics of sperm, the use of a variety of fluorescent probes was established. For sperm evaluation with fluorescent probes, a fluorescence microscope or flow cytometer is required. Although the fluorescence microscope is a cheaper option, the evaluation of spermatozoa by flow cytometry is recommended. This technique allows to evaluate about 10.000 sperm cells for several sperm features simultaneously (Cheuqueman et al., 2012), in a fast and accurate way. Assessment of sexed sperm morphology The assessment of sperm morphology by phase contrast microscopy is a very simple method to assess the effect of sexing on sperm morphology. Until recently, no effect of sexing process on percentage of cells with normal morphology had been reported (Carvalho et al., 2010). Because sexed sperm are submitted to a variety of adverse conditions during sorting, more detailed evaluation is needed. In this way, we used an atomic force microscope (AFM) to investigate if small changes could occur in the sperm head due to cell sorting (Carvalho et al., 2013). The AFM gives detailed three-dimensional information of a cell, with image of the surface of sperm at nanometer resolution (Berdyyeva et al., 2005). We acquired images from approximately 1800 sperm to measure 23 morphometric characteristics of the sexed and non sexed sperm head, such as volume, means radio, perimeter and surface area. Those measurements Anim. Reprod., v.11, n.3, p.199-206, Jul./Sept. 2014

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provided the features of Bos indicus sperm head (Fig. 1 A-C). The authors observed that non sexed sperm presented a higher minimum height, elongation and membrane roughness and a lower form factor, circularity ratio and degree of circularity than the sexed sperm. Moreover, simultaneous evaluation of all the measured features using discriminant analysis differentiated the sexed and the non sexed sperm with 100% accuracy. The differences in head shape of sexed and no sexed sperm may be related to modifications in

the plasma membrane, such as loss of some proteins and sperm capacitation. These modifications may cause decrease in longevity of the sperm in the female reproductive tract and compromise sperm binding in the oviduct to form the sperm reservoirs. Although AFM is an interesting and accurate tool for in vitro evaluation of the sperm head shape, their use as a routine is still limited due to the high cost of the equipment and the long time required acquiring the images.

Figure 1. Atomic force microscopy (AFM) 3D view images (A and B) and line profile (C), showing different dimensional parameters of the bovine sperm cells containing an X-chromosome. 1. Maximum diameter; 2. Width; 3. Perimeter; 4. Surface area; 5. Maximum height; 6. Average height.

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Effect of sexing process on sperm biochemical features Assessment of plasma membrane proteins Sperm plasma membrane is composed of protein, phospholipid, cholesterol and other components (Eddy and O’Brien, 1994). These proteins have an important function in protection and capacitation of sperm, as well as are necessary to fertilize the oocyte correctly. Therefore, any event that modifies the plasma membrane proteins, such as removing or cleaving these proteins can change the correct sperm function (Flesch and Gadella, 2000). It has been shown that intense manipulation of sperm during the sexing procedure, induced damage in plasma membrane and premature capacitation (Carvalho, 2013). Modification in protein profile of plasma membrane (McNutt and Johnson, 1996; Leahy et al., 2011), assessed by two-dimensional polyacrylamide gel electrophoresis has also being reported. According to McNutt and Johnson (1996), the sexing procedure can remove cleavage or change the glycosylation of membrane proteins. However, despite this evidence, it is not known exactly which proteins can be altered by the sexing process, or even if these proteins are directly related to the maintenance of sperm viability and the fertilization process. Because the membrane proteins have an important function, especially for events related to in vivo fertilization, it is possible that the lower fertility rate found when sexed sperm are used in vivo has correlation with modification on membrane protein profile of those sperm. Assess of methylation pattern As previously mentioned, assessment of DNA fragmentation has been the only method used for evaluating the effect of sexing on chromatin integrity (Blondin et al., 2009; Carvalho et al., 2010; Gosálvez et al., 2011). However, sperm DNA damage may result from DNA fragmentation, abnormal chromatin packaging and epigenetic defects (Tavalaee et al., 2009). DNA methylation is the most well characterized example of epigenetic contribution of the sperm nucleus to the developing embryo (Carrel and Hammound, 2010). In addition, changes in DNA methylation can alter regulation of gene expression (Bird, 2002; Jaenisch and Bird, 2003). Changes in methylation pattern of two important imprinted genes, insulin-like growth factor 2 (IGF2) and insulin-like growth factor-2 receptor (IGF2R), has been related to assisted reproduction technologies (ARTs), and may produce problems of embryo development and placentation (Curchoe et al., 2005; Long and Cai, 2007). To investigate the effect of sexing process in sperm methylation, Carvalho et al. (2012), using the quantitative bisulfite sequencing method, evaluated the methylation of distinct regions of 202

the IGF2 and IGF2R gene for non sexed, sexed for Xsperm and sexed for Y-sperm. The authors have not found changes in methylation pattern between the different groups evaluated for both genes (Fig. 2). Although both genes evaluated in that study are imprinted, it is not known whether these regions are packaged by histones or protamines, making these regions more or less susceptible to changes. Moreover, it is important to point out that we only assessed two regions of the genome, and it cannot be assumed that other regions do not have altered patterns of methylation due to sexing. It should also be considered that epigenetic changes may be related not only to changes in the DNA, but also to histone modifications. Therefore, an assessment of other genes or a new approach allowing an evaluation of a larger number of genes in sexed sperm, as well as studies of the effect of sexing process to another epigenetic mechanisms, could demonstrate if the sex-sorting affect other sperm epigenetic characteristics. Assessment of the longevity of sexed sperm Currently, changes in IVF protocols, such as sperm preparation and co-incubation time between sperm and oocyte, have been used to increase the blastocyst rate when sexed sperm are used (Blondin et al., 2009; Carvalho et al., 2010; Villamil et al., 2012). However, the reduced fertility rates after AI or embryo transfer program (Seidel et al., 1999; Sartori et al., 2004; Bodmer et al., 2005; Andersson et al., 2006; Peippo et al., 2009; Dejanette et al., 2010, 2011; Mellado et al., 2010; Underwood et al., 2010a, b; Sales et al., 2011; Healy et al., 2013) remain a problem for the use of sexed sperm in vivo. These results associated with the differences between in vitro and in vivo conditions necessary for fertilization suggest that the sexing process can compromise parameters that, although are not important to in vitro fertilization, could be essential for in vivo fertilization. Among them, the time of sperm survival on the female reproductive tract can be highlighted. A study by Ijaz et al. (1994) showed that the sperm can remain viable up to 30 h after thawing. Therefore, assessment of sperm characteristics, such as motility, plasma membrane and acrosome, over different times of incubation (Mocé et al., 2006; Carvalho, 2013), could identify the effect of sperm sexing on longevity. In a recent study (Carvalho, 2013), using flow cytometry to assess several sperm characteristics, we showed that the sexing process has negative effect on motility, mitochondrial membrane potential and integrity of plasma and acrosome membrane. Moreover, it was identified higher level of plasma membrane destabilization and less mitochondrial membrane potential of sexed sperm. Such difference was kept even at 12 h of incubation at 3°C in 5% CO2 in air. Although those studies do not reflect the actual condition of spermatozoa in the female Anim. Reprod., v.11, n.3, p.199-206, Jul./Sept. 2014

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reproductive tract, this is an indication that sexed sperm have lower resistance, and subsequently remain viable for a shorter period of time after thawing than the non sexed sperm. Considering the differences found in the viability of sexed and non sexed sperm, it could be suggested that increasing sperm concentration in the dose used for AI could compensate the reduced quality of sexed sperm, increasing their in vivo fertility. However,

Dejarnette et al. (2011), using 2.1 or 10 x 106 sperm per dose of sexed or non sexed sperm in AI, observed that the non sexed sperm had higher conception rates than sexed sperm, regardless the concentration used. According to these authors, factors other than concentration may be responsible for the lower conception rates obtained when sexed sperm is used. One of these factors could be the sperm ability to bind to oviduct epithelial cells, to form the sperm reservoir.

Figure 2. Methylation patterns in the DMR of the last exon of the IGF2 gene (A-C) and the second imprinting control region (ICR2) of the IGF2R gene (D-F) in non-sexed (A and D), sexed X (B and E) and sexed Y sperm (C and F) from four Nellore bulls. The arrow indicates the very specific methylation patterns observed in the 25th and 26th CpG sites, which had high methylation. White and dark circles represent unmethylated and methylated CpGs, respectively; horizontal lines of circles represent one clone, and the number of clones with the same methylation patterns is indicated at the right end of the lines. The data are the average of four bulls (three replicates per bull for the IGF2 gene and two replicates per bull for the IGF2R gene. Assessment of sexed sperm ability to bind to oviduct epithelial explants The oviduct sperm reservoir regulates the timing of sperm capacitation (Fazeli et al., 1999; Tienthai et al., 2004), helps to maintain sperm viability in female reproductive tract, and synchronizes the release of a fertile sperm with ovulation (Pollard et al., 1991). The formation of the sperm reservoir is dependent on the presence of sugars and proteins in sperm membrane (Green et al., 2001; Gwathmey et al., 2003; Foye-Jackson et al., 2011; Kadirvel et al., 2012). Therefore, the beginning of capacitation (Carvalho, 2013), as well as changes in the protein profile of the plasma membrane after sexing process could alter the Anim. Reprod., v.11, n.3, p.199-206, Jul./Sept. 2014

sperm binding to the oviduct cells. Thereby, it is possible that the lower in vivo fertility obtained when sexed sperm is used, could be related to changes in the formation and release of the sexed sperm from sperm reservoir. Based on that, Carvalho (2013), using epithelial oviduct explants, assessed the capacity of the sexed sperm to bind to oviduct cells after 30 min and 24 h of co-incubation. The number of sperm bound per mm of the explants was similar between sexed and non sexed sperm after 30 min (67.1 ± 9.0 and 70.3 ± 8.0, respectively) of co-incubation. However, after 24 h of co-incubation, there were less sexed sperm (6.7 ± 2.0) bound per mm of oviduct explants than non sexed sperm (23.6 ± 7.2). This suggests that sexed sperm has the ability to bind and to form the oviduct reservoirs, but 203

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has a reduced ability to remain attached to the reservoirs compared to non sexed sperm. This could be responsible by the lower in vivo fertility of sexed sperm since the higher number of the sperm bound to the explants after 24 h of co-incubation has positive correlation with cattle non-return rate (De Pauw et al., 2002). Another aspect to be considered is the evidence that a correct sperm-oviduct cell communication is needed to ensure the correct environment for fertilization and early embryonic development, since this interaction induces changes in gene expression (Fazeli et al., 2004; Kodithuwakku et al., 2007; FoyeJackson et al., 2011) and protein secretion by those cells (Georgiou et al., 2005, 2007). Therefore, changes in the sperm reservoir, could compromise the oviduct environment, with higher number of unfertilized oocytes (Sartori et al., 2004; Schenk et al., 2006; Peippo et al., 2009) or embryos with low quality (Schenk et al., 2006; Peippo et al., 2009; Larson et al., 2010) reported when sexed sperm was used. Final consideration This review presented the most recent results related to the effect of sexing in the structure and function of bovine sexed sperm. Although several studies have already evaluated the effect of sexing process in structural and functional characteristics of sperm, the real cause of lower fertility of those sperm, especially when used in vivo has not yet been identified. Possibly, the lower fertility of sexed sperm has a multifactorial cause, since the sperm are complex cells that need the integrity and functionality of multiple attributes to successfully fertilize the oocyte. Regarding changes in sperm caused by the sexing process, discussed in this review, we can list altered sperm motility, membrane integrity and acrosome, premature capacitation with modification of their membrane proteins and less ability to remain viable after thawing. This reduced viability of sexed sperm observed after thawing is responsible for the shorter longevity and consequently, less ability to remain bound to the oviduct cells after formation of sperm reservoirs. Those information lead us to suggest that the best moment for AI using sexed sperm is near ovulation time. This delay in the moment of AI may reduce the waiting time of the sperm in the female reproductive tract, ensuring larger numbers of viable cells at the time of ovulation. Ackowledgments The authors thank FAPESP, CNPq, CAPE and EMBRAPA for financial support. References Alomar M, Mahieu J, Verhaeghe B, Defoin L, Donnay I. 2006. Assessment of sperm quality parameters of six bulls showing different abilities to 204

promote embryo development in vitro. Reprod Fertil Dev, 18:395-402. Amann RP, Hammerstedt RH. 1993. In vitro evaluation of sperm quality: an opinion. J Androl, 14:397-406. Andersson M, Taponen J, Kommeri M, Dahlbom M. 2006. Pregnancy rates in lactating Holstein-Friesian cows after artificial insemination with sexed sperm. Reprod Domest Anim, 41:95-97. Balhorn R, Gledhill BL, Wyrobek AJ. 1977. Mouse sperm chromatin proteins: quantitative isolation and partial characterization. Biochemistry, 16:4074-4080. Berdyyeva T, Woodworth CD, Sokolov I. 2005. Visualization of cytoskeletal elements by the atomic force microscope. Ultramicroscopy, 102:189-198. Bird A. 2002. DNA methylation patterns and epigenetic memory. Genes Dev, 16:6-21. Blondin P, Beaulieu M, Fournier V, Morin N, Crawford L, Madan P, King WA. 2009. Analysis of bovine sexed sperm for IVF from sorting to the embryo. Theriogenology, 71:30-38. Bodmer M, Janett F, Hassig M, den Daas N, Reichert P, Thun R. 2005. Fertility in heifers and cows after low dose insemination with sex-sorted and nonsorted sperm under field conditions. Theriogenology, 64:1647-1655. Brito, LFC, Barth AD, Bilodeau-goeseels S, Panich PL, Kastelic JP. 2003. Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate. Theriogenology, 60:1539-1551. Butler ST, Hutchinson IA, Cromie AR, Shalloo L. 2014. Applications and cost benefits of sexed semen in pasture-based dairy production systems. Animal, 8:165172. Carrel DT, Hammound SS. 2010. The human sperm epigenome and its potential role in embryonic development. Mol Hum Reprod, 16:37-47. Carvalho JO, Sartori R, Lemes AP, Mourão GB, Dode MAN. 2009. Cinética de espermatozoides criopreservados de bovino após sexagem por citometria de fluxo. Pesq Agropec Bras, 10:1346:1351. Carvalho JO, Sartori R, Machado GM, Mourao GB, Dode MA. 2010. Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production. Theriogenology, 74:1521-1530. Carvalho JO, Michalczechen-Lacerda VA, Sartori R, Rodrigues FC, Bravin O, Franco MM, Dode MAN. 2012. The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting. Mol Reprod Dev, 79:77-84. Carvalho JO. 2013. Aspectos moleculares, estruturais e funcionais de espermatozoides bovinos sexados por citometria de fluxo. São Paulo, SP: Universidade de São Paulo. Tese de Doutorado. Carvalho JO, Silva L, Sartori R. Dode MAN. 2013. Nanoscale differences in the shape and size of x and y Anim. Reprod., v.11, n.3, p.199-206, Jul./Sept. 2014

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Georgiou AS, Sostaric E, Wong CH, Snijders AP, Wright PC, Moore HD, Fazeli A. 2005. Gametes alter the oviductal secretory proteome. Mol Cell Proteomics, 4:1785-1796. Georgiou AS, Snijders AP, Sostaric E, Aflatoonian R, Vazquez JL, Vazquez JM, Roca J, Martinez EA, Wright PC, Fazeli A. 2007. Modulation of the oviductal environment by gametes. J Proteome Res, 6:4656-4666. Gosálvez J, Ramirez MA, Lopez-Fernandez C, Crespo F, Evans KM, Kjelland ME, Moreno JF. 2011. Sex-sorted bovine spermatozoa and DNA damage: I. Static features. Theriogenology, 75:197-205. Green CE, Bredl J, Holt WV, Watson PF, Fazeli A. 2001. Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro. Reproduction, 122:305-315. Gwathmey TM, Ignotz GG, Suarez SS. 2003. PDC109 (BSP-A1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir. Biol Reprod, 69:809-815. Healy AA, House JK, Thonson PC. 2013. Artificial insemination field data on the use of sexed and conventional semen in nulliparous Holstein heifers. J Dairy Sci, 96:1905-1914. Hollinshead FK, O’Brien, JK Maxwell WMC, Evans G. 2004. Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa. Theriogenology, 62:958-968. Ijaz A, Lambert RD, Sirard MA. 1994. In vitro cultured bovine granulosa and oviductal cells secrete sperm motility-maintaining factors. Mol Reprod Dev, 37:54-60. Jaenisch R, Bird A. 2003. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet, 33:245-254. Johnson LA, Flook JP, Look MV, Pinkel D. 1987. Flow sorting of X and Y chromosome bearing into two populations. Gamete Res, 16:1-9. Kadirvel G, Machado SA, Korneli C, Collins E, Miller P, Bess KN, Aoki K, Tiemeyer M, Bovin N, Miller DJ. 2012. Porcine sperm bind to specific 6sialylated biantennary glycans to form the oviduct reservoir. Biol Reprod, 87:147. Kaneko S, Oshio S, Kobayashi T, Lizuka R, Mohri H. 1984. Human X and Y bearing sperm differ in cell surface sialic acid content. Biochem Biophys Res, 124:950-955. Klinc P, Rath D. 2007. Reduction of oxidative stress in bovine spermatozoa during flow cytometric sorting. Reprod Domest Anim, 42:63-67. Kodithuwakku SP, Miyamoto A, Wijayagunawardane MP. 2007. Spermatozoa stimulate prostaglandin synthesis and secretion in bovine oviductal epithelial cells. Reproduction, 133:1087-1094. Koo CG, Stackpole CW, Boyse EA, Hämmerling U. 1973. Topographical, localization of the H-Y antigen on 205

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mouse spermatozoa by immunoelectron microscopy. Proc Natl Acad Sci USA, 70:1502-1505. Larson JE, Lamb GC, Funnell BJ, Bird S, Martins A, Rodgers JC. 2010. Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen. Theriogenology, 73:698-703. Leahy T, Marti JI, Crossett B, Evan G, Maxwell WMC. 2011. Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm. Theriogenology, 75:962-971. Long JE, Cai X. 2007. Igf-2r expression regulated by epigenetic modification and the locus of gene imprinting disrupted in cloned cattle. Gene, 388:125-134. Malmgren L. 1997. Assessing the quality of raw semen: a review. Theriogenology, 48:523-530. McNutt TL, Johnson LA. 1996. Electrophoretic gel analysis of Hoechst 33342 stained and flow cytometrically sorted bovine sperm membrane proteins. Reprod Domest Anim, 31:703-709. Mellado M, Coronel F, Estrada A, Rios FG. 2010. Fertility in Holstein x Gyr cows in a subtropical environment after insemination with Gyr sex-sorted semen. Trop Anim Health Prod, 42:1493-1496. Mocé E, Graham JK, Schenk JL. 2006. Effect of sexsorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction. Theriogenology, 66:929-936. Peippo J, Vartia K, Kananen-Anttila K, Raty M, Korhonen K, Hurme T, Myllymaki H, Sairanen A, Maki-Tanila A. 2009. Embryo production from superovulated Holstein-Friesian dairy heifers and cows after insemination with frozen-thawed sex-sorted X spermatozoa or unsorted semen. Anim Reprod Sci, 111:80-92. Pollard JW, Plante C King WA, Hansen PJ, Betteridge KJ, Suarez SS. 1991. Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells. Biol Reprod, 44:102-107. Sales JN, Neves KA, Souza AH, Crepaldi GA, Sala RV, Fosado M, Campos Filho EP, Faria M, Sá Filho MF, Baruselli PS. 2011. Timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm. Theriogenology, 76:427-435. Sartori R, Souza AH, Guenther JN, Caraviello DZ, Schenk JL, Wiltbank MC. 2004. Fertilization rate and embryo quality in superovulated Holstein heifers artificially inseminated with X-sorted or unsorted sperm. Anim Reprod, 1:86-90. Schenk JL, Suh TK, Seidel Jr GE. 2006. Embryo production from superovulated cattle following insemination of sexed sperm. Theriogenology, 65:299307. Schenk JL, Cran DG, Everett RW, Seidel Jr GE. 2009. Pregnancy rates in heifers and cows with

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cryopreserved sexed sperm: Effects of sperm numbers per inseminate, sorting pressure and sperm storage before sorting. Theriogenology, 71:717-728. Seidel GE Jr, Schenk JL, Herickhoff LA, Doyle SP, Brink Z, Green RD, Cran DG. 1999. Insemination of heifers with sexed sperm. Theriogenology, 52:14071420. Seidel GE Jr, Garner DL. 2002. Current status of sexing mammalian sperm. Reproduction, 124:733-743. Sharpe JC, Evans KM. 2009. Advances in flow cytometry for sperm sexing. Theriogenology, 71:4-10. Smith LC. 1993. Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro. J Reprod Fertil, 99:39-34. Soon LL, Ausio J, Breed WG, Power JH, Muller S. 1997. Isolation of histones and related chromatin structure from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata. J Exp Zool, 278:322-332 Spinaci, M, Bucci D, Chlapanidas T, Vallorani C, Perteghella S, Communod R, Vigo D, Tamanini C, Galeati G, Faustini M, Torre ML. 2013. Boar sperm changes after sorting and encapsulation in barium alginate membranes. Theriogenology, 80:526-532. Suh TK, Schenk JL, Seidel Jr GE. 2005. High pressure flow cytometric sorting damages sperm. Theriogenology, 64:1035-1048. Tavalaee M, Razavi S, Nasr-Esfahani MH. 2009. Influence of sperm chromatin anomalies on assisted reproductive technology outcome. Fertil Steril, 91:1119-1126. Tienthai P, Johannisson A Rodriguez-Martinez H. 2004. Sperm capacitation in the porcine oviduct. Anim Reprod Sci, 80:131-146. Underwood SL, Bathgate R, Ebsworth M, Maxwell WM, Evans G. 2010a. Pregnancy loss in heifers after artificial insemination with frozen-thawed, sex-sorted, re-frozen-thawed dairy bull sperm. Anim Reprod Sci, 118:7-12. Underwood SL, Bathgate R, Maxwell WM, Evans G. 2010b. Birth of offspring after artificial insemination of heifers with frozen-thawed, sex-sorted, re-frozenthawed bull sperm. Anim Reprod Sci, 118:171-175. Villamil RP, Wel H, Moreira G, Caccia M, Fernandez Taranco M, Bó GA. 2012. Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll. Theriogenology, 78:165-171. Watkins AM, Chan PJ, Kalugdan TH, Patton WC, Jacobson JD, King A. 1996. Analysis of the flow cytometer stain Hoechst 33342 on human spermatozoa. Mol Hum Reprod, 2:709-712. Wheeler BW, Rutledge JJ, Fischer-Brown A, Vanetten T, Malusky S, Beebe DJ. 2006. Application of sexed semen technology to in vitro embryo production in cattle. Theriogenology, 65:219-227.

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Advances in reproductive management: pregnancy diagnosis in ruminants T.L. Ott1, C. Dechow, M.L. O’Connor Department of Animal Science, Pennsylvania State University, University Park, PA, USA.

Abstract During the last 10 years the U.S. dairy industry has experienced a reversal of the decades-long trend in declining fertility traits. In fact, there is evidence that, nationally, this is contributing to improvements in pregnancy rates. And while these measures are still close to their historical lows, there is reason for optimism that this reversal will continue into the future. The reasons for improved pregnancy rates are related to use of biotechnologies and improved management practices for high producing dairy cows as well as greater emphasis on genetic selection for fertility-related traits. Combined, these factors have resulted in a reduction in the average days to first service in our national dairy herd of approximately 10 days over the past decade and a reduction in calving interval of approximately 15 days. However, current challenges include accurate identification of cows that fail to conceive following insemination and their timely reinsemination. The primary metric for success of pregnancy diagnosis is the inter-service interval, or the number of days between insemination and the subsequent insemination in a cow that fails to conceive or that loses an established pregnancy. This trait is directly affected by the choice of pregnancy diagnosis method. Pregnancy diagnosis methods include estrous detection (visual or assisted), transrectal palpation of uterine contents, transrectal ultrasound visualization of uterine contents and assay for hormones in blood, milk or other body fluids. Each of these methods has advantages and disadvantages. Presently, ultrasound and blood hormone assay at 28 days after insemination offer the earliest specific diagnostics for determining pregnancy status. However, other methods are on the horizon that may provide opportunities to further reduce the interval between insemination and accurate diagnosis of pregnancy status of dairy cattle. One of these targets identification of failed inseminations 18 to 20 days after insemination. This approach, if successful, would allow identification of a portion of open cows prior to their expected return to estrus. The ultimate goal is to identify cows that fail to conceive to an insemination in time to reinseminate them at a normal cycle interval (21 to 23 days) while achieving high

_________________________________________ 1 Corresponding author: [email protected] Phone: +1(814)865-5989 Received: June 7, 2014-07-09 Accepted: July 15, 2014

conception rates. Reproductive management programs that utilize early pregnancy diagnosis will reduce the interservice interval and improve pregnancy rate, which is a key metric in determining profitability on dairy farms. Keywords: cattle, diagnostic, fertility, interferon, pregnancy, ultrasound. Introduction Poor reproductive performance remains one of the primary reasons for involuntary culling of dairy cows in the U.S. and globally. Roughly one third of dairy cows culled annually in the U.S. are culled due to reproductive problems (DeVries et al., 2010; Pinedo and DeVries, 2010). Lactating cows that fail to conceive are eventually culled for low production late in lactation at high cost to the dairy farmer (Britt, 1985). Low fertility results in reduced herd milk production, increased cost associated with multiple inseminations and increased number of replacement heifers needed to maintain herd size (Britt, 1985). Fortunately, during the last 10 years the U.S. dairy industry has experienced a reversal of the decades-long trend in declining fertility traits. In fact, trends in fertility traits are increasing in the U.S. (Fig. 1). And while fertility traits are still close to their historic lows, there is reason for optimism that this reversal will continue into the future. Pregnancy rate is the product of the estrous detection or submission rate (for dairies using timed artificial insemination) and the conception rate. It is a measure of how quickly cows that are eligible to become pregnant (i.e. after the voluntary waiting period) actually become pregnant. Improved fertility traits and use of technologies to improve submission rates have been largely responsible for the improved pregnancy rates during the last decade. For example, use of ovulation synchronization (e.g. Ovsynch) with timed artificial insemination (TAI) has resulted in more cows getting their first postpartum insemination closer to the end of the voluntary waiting period. One current challenge, however, is early and accurate diagnosis of pregnancy status to allow for cows that failed to conceive or maintain pregnancy to be reinseminated in a timely fashion.

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Genetic Trends for Productive Life in U.S. Dairy Cows 4 3

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Figure 1. A) Genetic trends for cow and sire breeding value (BV) for productive life in U.S. dairy cows, and B) Genetic trends for daughter pregnancy rate in U.S. Dairy cows. From Council for Dairy Cattle Breeding, 2014.

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Trends in genetic selection for fertility traits Since its inception 20 years ago, the Lifetime Net Merit index (NM$) in the U.S. has increased genetic selection for fertility- and health-related traits. In 1994, when Net Merit Dollar index was introduced it was weighted roughly 75% for production traits (yield, fat and protein). Today’s NM$ gives only ~35% weight to production traits with the remaining emphasis placed on health and fitness traits. These include productive life (PL; 22% current weight), which was introduced in 1994 and daughter pregnancy rate (DPR) which was introduced in 2003 with a weight of 6% and increased twice to its present weight in the index of 11%. While it will take some time to determine the full effects of these latest changes, it is clear they have resulted in improved genetic merit for fertility traits in the U.S. dairy herd. Currently, the difference in predicted transmitting ability (PTA) between the highest and lowest ranking bulls for DPR is approximately 8, which translates to daughters of the highest ranking DPR bulls conceiving, on average, 32 days sooner than those from the lowest ranking DPR bulls. Furthermore, with the routine collection of more fertility related data (e.g. cow conception rate, heifer conception rate) along with other health and fitness traits, it is likely that we will see continued evolution of NM$ towards more robust selection of fertility and health traits (Dechow, 2014). Trends in reproductive management strategies During this same 20 year span, approaches to reproductive management have also changed. In 1994, the majority of dairies bred cows based on estrous detection with few farms using estrous synchronization (Miller et al., 2007). With the advent of ovulation synchronization programs in the mid-1990’s, farms that were struggling with estrous detection had another tool to manage reproduction (Pursley et al., 1995, 1997). This period was also accompanied by increased use of transrectal ultrasound and blood and milk hormone tests for pregnancy diagnosis. Ultrasound evaluation of ovarian structures also increased the ability to tailor reproductive management to ovarian status. Today, most dairies use a combination of insemination based on estrous detection and synchronization of ovulation coupled with TAI (Caraviello et al., 2006). Combining these approaches is the most economical way to improve pregnancy rates given typical rates of synchronization drug injection compliance and estrous detection efficiencies (Galvão et al., 2013). Fewer dairies are choosing to use on-farm bulls for mating cows, and for good reason. Bull breeding should be considered a choice of last resort for modern dairies. Aside from their lower genetic merit, on-farm bulls consume feed and occupy facilities, suffer from infertility and venereal diseases, require veterinary Anim. Reprod., v.11, n.3, p.207-216, Jul./Sept. 2014

attention, and cause injuries and deaths on farms each year (Lima et al., 2010). Efficiency and accuracy of estrous detection have also benefited from development of tools including simple tail head chalking/painting and glue-on mount detectors to higher tech pedometers and activity monitors containing accelerometers that continuously monitor a cow’s activity (Van Eerdenburg, 2008; Fricke et al., 2014a). Use of activity monitors with accelerometers will likely continue to increase because of their automation and compatibility with mobile devices and cloud-based data storage and analysis. This technology is particularly attractive for dairies of intermediate size (150 to 500 cows) that struggle to maximize efficiency of labor use (Fricke et al., 2014b) and for dairies that prefer not to use hormonal synchronization (Neves et al., 2012). For example, in a large study comparing the use of automated activity monitors (AAM) with synchronization and timed artificial insemination, AAM reduced days to first service in two of the three large dairies examined (Neves et al., 2012). Recently, comparison was made between AAM and a presynchronization-ovulation synchronization program with TAI (Stevenson et al., 2014). Interestingly, pregnancies per AI were modestly lower with insemination based on AAM, but cows became pregnant quicker with AAM compared to TAI, probably due to the earlier VWP in the AAM group (Stevenson et al., 2014). Together, estrous synchronization and ovulation synchronization with TAI are credited with reducing the average days to first service from 90 to 81 days over the last 10 years (Miller et al., 2007; Council on Dairy Cattle Breeding, 2014). This has resulted in a reduction of calving interval of about two weeks during this same period. In spite of these improvements in estrous detection technologies, low efficiency and accuracy of estrous detection remains the primary reason for dairies adopting ovulation synchronization and TAI programs (Goodling et al., 2005; Moore and Thatcher, 2006). Variations of the ovulation synchronization programs coupled with presynchronization (e.g. Presynch) provide a variety of options to fit producer needs. The Ovsynch 56 program (gonadotropin releasing hormone (GnRH) followed 7 days later by prostaglandin F2α (PGF), then a second GnRH injection 56 h later with TAI 16 h later), has proven to be the most effective ovulation synchronization program for maximizing pregnancy rates. This program is most effective when initiated at days 5 to 9 of the cows’ estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000). To accomplish this, presynchronization programs have been developed using either PGF (Presynch: two injections of PGF 14 days apart followed by Ovsynch 11 to 14 days later) or a combination Ovsynch without TAI followed by Ovsynch started 7 days later (Double-Ovsynch; Moreira et al., 2000; Souza et al., 2008). The advantage of the 209

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latter programs that include GnRH in the presynchronization is that cows that are not cycling will be induced to form a corpus luteum by use of GnRH (Herlihy et al., 2012; Ayres et al., 2013). In either case, use of presynchronization ensures that a greater number of cows will ovulate to the first GnRH of the ovulation synchronization program (Gumen et al., 2012; Ayres et al., 2013). Use of Presynch-Ovsynch programs on wellmanaged dairies yields pregnancy rates comparable to inseminating cows based on detected estrus (Rabiee et al., 2005; Stevenson et al., 2014), and a high percentage of large dairies in the U.S. use some form of hormonal synchronization with timed artificial insemination (Caraviello et al., 2006). Improvements in estrous detection and estrous and ovulation synchronization coupled with TAI have resulted in more cows being inseminated in the first 21 days after the voluntary waiting period. Improvements in fertility traits, while modest, should result in improved conceptions rates to these earlier services. Thus, improvements in both submission rates and conception rates will likely continue to yield slow, but steady improvement in pregnancy rates. The current challenge is to reduce the interservice interval for those cows that fail to conceive or that lose an established pregnancy. This challenge requires accurate and early detection of pregnancy status.

embryo when conducted at very early stages gestation (25 days in cattle); 2) presence of twins with increased accuracy; 3) fetal viability (e.g. heart beat); 4) fetal gender; and 5) ovarian structures (follicles and corpora lutea). Although many veterinarians now provide ultrasound service to dairies of all sizes, cost and frequency of veterinary visits is still limiting for smaller dairies.

Pregnancy diagnosis

Chemical pregnancy assays

Key to effective reproductive management programs is early and accurate pregnancy diagnosis following insemination. The goal is for cows to be reinseminated, on average, before 42 days after a failed insemination. Estrous detection following insemination remains a widely used approach to pregnancy diagnosis. However, dairies that struggle with accurate estrous detection often experience extended (>42 days) interservice intervals when relying on this method. In addition, inaccurate estrous detection increases the risk for insemination of pregnant cattle (Moore et al., 2005). Here again, use of second generation AAM may aid in detecting cows returning to estrus 21 to 24 days after insemination. Furthermore, for dairies that use ovulation synchronization and TAI for first services, some cows will not continue cycling after the first insemination and will not be detected in heat (Lucy et al., 2004).

Blood and milk progesterone assays have been available for over 20 years (O’Connor, 1994). However, because progesterone is not pregnancy-specific and the requirement for multiple assays to achieve acceptable specificity, these assays have not been widely adopted. However, development of sensitive automated inline milk progesterone assays should make this technology amenable to commercial application (Käppel et al., 2007; Fricke et al., 2014a). Automated inline testing should accelerate the adoption of progesterone assay for pregnancy diagnosis because of the high accuracy of sequential testing at insemination and then at 20 to 24 days after insemination for detecting failed inseminations (O’Connor, 1994). Testing at later intervals could then be used for confirming pregnancy status. Adoption of inline testing technology will likely take some time due to the high capital costs and will be dependent on the reliability of the automated inline tests. The first reliable pregnancy-specific hormone assays were developed to measure placenta-derived proteins. The first of these measured circulating concentrations of pregnancy-specific protein B (PSPB; Butler et al., 1982). Pregnancy-specific protein B is produced by placental giant binucleate cells that form from mononuclear trophoblast cells starting around days 17 to 19 of pregnancy in cattle (Spencer et al., 2007). Pregnancy-specific protein B concentrations begin to be

Transrectal palpation and ultrasound visualization of uterine contents Palpation of uterine contents per rectum is the most widely used method for pregnancy diagnosis in dairy cattle. The technique can be performed reliably after day 30 of pregnancy and is highly accurate when practiced by a skilled veterinarian or animal manager. Palpation does require training and experience to conduct with high accuracy and without damaging the 210

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reliably detectable in plasma starting at day 24, and by day 28 concentrations are sufficiently elevated to allow their use for a highly reliable test for pregnancy in ruminant animals (Sasser et al., 1986). Pregnancyspecific protein B is a member of the pregnancy associated glycoprotein (PAG) family of proteins which are encoded by a very large gene family (Xie et al., 1994). PAG are also secreted in milk and reliable milk PAG tests are now available in the U.S. for use after day 35 of early pregnancy (Green and Roberts, 2006). A number of commercial suppliers are now producing diagnostic tests for PAG family members in blood and milk and millions of samples are tested annually in the U.S. Currently, available PAG diagnostic tests require delivering samples to a centralized testing laboratory. Once these tests are adapted to inline milking systems and/or developed into “cow-side” diagnostics, they will be more widely adopted. New opportunities for early diagnosis of pregnancy status A large but ill-defined percentage of cows fail to conceive following insemination or lose embryos prior to rescuing CL function (Pereira et al., 2013). Theoretically, these cows could be detected in estrus and be reinseminated 21 to 24 days after their first insemination. However, as stated above, not all these cows will exhibit estrous behavior and those that do often go undetected resulting in less than 50% of open cows being detected in estrus 21 to 24 days after insemination. These cows have been called “phantom” cows (Lucy et al., 2004). Therefore, if a pregnancyspecific signal could be detected during early pregnancy it could be used to identify failed conceptions and allow for reinsemination of open cows at 21 to 24 day intervals. Interferon tau (IFN-τ) is the conceptus signal responsible for rescuing CL function in ruminants (Bazer et al., 2009). With its discovery and characterization, a number of groups attempted to detect IFN-τ in systemic circulation as a method for determining conceptus signaling during early pregnancy (Stewart et al., 1992). With the exception of one study with a small number of pregnant sheep (Schalue-Francis et al., 1991), the outcomes of a number of studies supported the prevailing hypothesis that IFN-τ did not escape the uterus in appreciable quantities. It was generally accepted that IFN-τ acted locally on the uterine endometrium to alter the pattern of PGF release and maintain CL function (Spencer and Bazer, 2004). This is in contrast to humans where conceptus-produced chorionic gonadotropin directly supports CL function and can be measured in maternal blood and urine as soon as 6 to7 days following fertilization (Bazer et al., 1991). Early studies either used antiviral assay for detecting interferon activity (Pontzer et al., 1988) or RIA (Vallet et al., 1988) or ELISA (Zhu et al., 1996) to Anim. Reprod., v.11, n.3, p.207-216, Jul./Sept. 2014

directly assay for IFN-τ. More recently, we addressed the question of systemic responses to conceptus signaling in ruminants using a different, indirect, approach of assaying for expression of interferon stimulated genes (ISG) in peripheral blood leukocytes (Yankey et al., 2001). Type I interferons such as IFN-τ induce a large number of ISG that are better known in the immune response to viral infection (Williams, 1991). Among these are the myxovirus resistance genes (MX1 and MX2; Ott et al., 1998) and interferon stimulated gene 15 (ISG15; Austin et al., 2004). Results of Yankey et al. (2001) were the first to show that early pregnancy resulted in increased abundance of mRNA and protein for MX1 in peripheral blood leukocytes (PBL) of pregnant compared to non-pregnant ewes on day 15 after insemination (Fig. 2). The effect of early pregnancy signaling on ISG expression in PBL was subsequently confirmed in cattle (Han et al., 2006; Gifford et al., 2007; Stevenson et al., 2007; Oliveira et al., 2008; Green et al., 2010; Ribeiro et al., 2014). Figure 3 shows abundance of mRNA for MX2 and ISG15 in PBL of dairy cattle following insemination (Gifford et al., 2007). Messenger RNA for ISG15 was increased in PBL of pregnant compared to bred, nonpregnant, dairy cows on day 18 and 20 after insemination and MX2 mRNA abundance was greater at day 16, 18 and 20 after insemination. Importantly, differences in expression of ISG occurred prior to the expected time of return to estrus (day 21 to 24). These results caused a careful reevaluation of hypothesis that IFN-τ acted solely in a paracrine fashion to alter uterine PGF production and maintain CL function (Oliveira et al., 2008). Consistent with previous results, Oliveira et al. (2008) demonstrated that expression of interferon stimulated genes increased in the peripheral blood of pregnant sheep at day 15. Furthermore, they showed that antiviral activity (a measure of IFN) was elevated in uterine vein plasma, but not plasma obtained from the uterine artery during early pregnancy. These results strongly suggested that there was endocrine release of IFN-τ or a related IFN from the uterus in response to conceptus IFN-τ. This hypothesis was confirmed in subsequent studies using implanted mini-pumps delivering low doses of IFN-τ into the uterine vein that protected the CL from a subluteolytic dose of PGF (Bott et al., 2010; Antoniazzi et al., 2013). These experiments strongly supported the hypothesis that IFN-τ exits the uterus, induces ISG in blood cells and peripheral tissues including CL and liver and that this mechanism may contribute to protecting the CL from the luteolytic effects of PGF (Antoniazzi et al., 2013). From a practical standpoint, the fact that the presence of a conceptus induces ISG in peripheral blood prior to expected return to estrus provides an opportunity to detect failed inseminations during a period when the ovary should contain a second or third wave dominant follicle that could be induced to ovulate. This would allow reinsemination of open cows at 21 to 211

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24 day intervals (Lucy et al., 2004). In this model, cows detected open at day 18 to 20 after insemination would receive injection of a luteolytic dose of PGF and be bred on detected estrus or receive TAI accompanied by a GnRH injection 48 to72 h after the PGF injection (Lucy et al., 2004). The effectiveness of such a program for reinsemination of open cows has not yet been determined. It would be highly dependent on the percentage of open cows that are detectable at 18 to 20 days after insemination, which has not been determined in replicated large-scale studies. However, estimates are that greater than 50% of failed inseminations have occurred by 20 days after insemination (Pereira et al., 2013). Any diagnostic test developed for this purpose would need to exhibit a high degree of specificity because it would trigger a management decision to lyse the CL with PGF and return the cow to estrus. Of course, cows with elevated expression of ISG at this time would be presumed pregnant, but pregnancy would need to be confirmed later in gestation to account for later embryo losses. A diagnostic used as described here could not be considered a pregnancy test, due to the relatively high degree of embryo loss that occurs between day 20 and 45 after insemination (Pereira et al., 2013). The usefulness of such a diagnostic would be for detecting open cows. Finally, studies on conceptus-uterus-immune cell cross-talk during early pregnancy have raised new questions related to the function of these ISG both locally in the uterus and in the peripheral tissues

during early pregnancy (Ott and Gifford, 2010). Interferon stimulated genes generally function as part of the innate immune response to viral infection. For example, MX1 blocks the replication of negativestranded RNA viruses by interfering with generation of viral transcripts or by inhibiting assembly of mature viral particles depending on the species of animal (Haller and Kochs, 2011). Whether activation of ISG in early pregnancy is to elevate innate immunity (during a period when some aspects of immune function are down regulated to protect the allogeneic conceptus) is a question currently under investigation (Ott and Gifford, 2010). Furthermore, assaying for expression of interferon stimulated genes in blood during early pregnancy provides a non-invasive window on conceptus-uterine cross-talk during early pregnancy (Gifford et al., 2008). For example, Ribeiro et al. (2014) recently showed that treatment of lactating cows with sequential low doses of recombinant bovine somatotropin 14 days apart starting at insemination improved conceptus growth and pregnancy rates (Ribeiro et al., 2014). Enhanced conceptus growth was also reflected in increased abundance of ISG15 mRNA in peripheral blood leukocytes at day 19 after insemination in cows that maintained their pregnancies (Fig. 4). Interestingly, not all ISG responded in a similar fashion suggesting that much more remains to be learned about conceptus-uterus-immune cell cross-talk during early pregnancy in ruminants (Ribeiro et al., 2014).

600

Mx mRNA (CNTS/mm2)

500 400 300

200 100 0 0

9

12

15

18 21 Days post - AI

24

27

30

Figure 2. Steady state abundance of myxovirus resistance 1 (Mx) mRNA in peripheral blood lymphocytes of pregnant (black bars) and bred, non-pregnant (grey bars) ewes from insemination (day 0) to 30 days after insemination. Mx mRNA was increased in pregnant ewes from day 15 to 30 after insemination. From Yankey et al., 2001. 212

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Mx2 Relative fold change

6 4 2 0

16

18 Days after insemination

20

ISG-15

Relative fold change

6 4 2 0

16

18 20 Days after insemination

Figure 3. Relative fold change in steady state abundance of mRNA for interferon stimulated gene 15 (ISG-15) and myxovirus resistance 2 (Mx2) in peripheral blood leukocytes at 16, 18 and 20 days after insemination in pregnant (filled bars) and bred, non-pregnant dairy cows (open bars). *indicates statistical difference between pregnancy statuses (P < 0.05). From Gifford et al., 2007.

7 Relative mRNA expression

ISG15

c

6 5 4

2

a

a

3 b

b

b

1 0

Control S-bST T-bST Figure 4. Effects of a single low dose (325 mg) of bovine somatotropin (S-bST) given at insemination or two sequential S-bST at insemination and 14 days later on relative mRNA abundance of ISG15 at day 19 after insemination in cows diagnosed pregnant (open bars) and open (filled bars) 31 days after insemination. From Ribeiro et al., 2014.

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Conclusions Obtaining optimal pregnancy rates is a key to success in the dairy business. Technologies developed over the last 25 years have improved the ability of producers to accurately detect estrus and monitor pregnancy status while maximizing labor efficiency. Coupled with an increase focus on reproductive and fitness traits in selection indexes, dairy producers are beginning to reverse the decades-long trend in declining reproductive performance in the U.S. Automated continuous activity monitors containing accelerometers will allow real-time evaluation of health status and improve estrous detection rates. Integration of these systems with cloud computing and mobile device communication will provide producers with continuous information about the status of cows in the herd. The challenge will be with handling large data sets and distilling down information in a form that is useful to support on-farm decision making. There will likely be increased adoption of pregnancy-specific hormone assays, especially if these assays can be adapted to inline or “cow-side” diagnostic platforms as has been done for the milk progesterone assay. Ultrasound will remain the gold-standard for evaluating reproductive status and affordability and ease of use are likely to continue to improve. Optimized hormonal synchronization protocols currently allow producers to precisely target first inseminations. However, detection of failed inseminations and timely reinsemination of cows continues to be a challenge that increases days open and reduces profitability. New diagnostic approaches are targeting detection of open cows 18 to 20 days after insemination. If successful, they should allow a large proportion of open cows to be identified and reinseminated at 21 to 24 day intervals. References Antoniazzi AQ, Webb BT, Romero JJ, Ashley RL, Smirnova NP, Henkes LE, Bott RC, Oliveira JF, Niswender GD, Bazer FW, Hansen TR. 2013. Endocrine delivery of interferon tau protects the corpus luteum from prostaglandin F2 alpha-induced luteolysis in ewes. Biol Reprod, 88:1-12. Austin KJ, Carr AL, Pru JK, Hearne CE, George EL, Belden EL, Hansen TR. 2004. Localization of ISG15 and conjugated proteins in bovine endometrium using immunohistochemistry and electron microscopy. Endocrinology,145:967-975. Ayres H, Ferreira RM, Cunha AP, Araújo RR, Wiltbank MC. 2013. Double-Ovsynch in highproducing dairy cows: effects on progesterone concentrations and ovulation to GnRH treatments. Theriogenology, 79:159-164. Bazer FW, Simmen RC, Simmen FA. 1991. Comparative aspects of conceptus signals for maternal recognition of pregnancy. Ann NY Acad Sci, 622:202214

211. Bazer FW, Spencer TE, Johnson GA, Burghardt RC, Wu G. 2009. Comparative aspects of implantation. Reproduction, 138:195-209. Bott RC, Ashley RL, Henkes LE, Antoniazzi AQ, Bruemmer JE, Niswender GD, Bazer FW, Spencer TE, Smirnova NP, Anthony RV, Hansen TR. 2010. Uterine vein infusion of interferon tau (IFNT) extends luteal life span in ewes. Biol Reprod, 82:725-735. Britt JH. 1985. Enhanced reproduction and its economic implications. J Dairy Sci, 68:1585-1592. Butler JE, Hamilton WC, Sasser RG, Ruder CA, Hass GM, Williams RJ. 1982. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol Reprod, 26:925-933. Caraviello DZ, Weigel KA, Fricke PM, Wiltbank MC, Florent MJ, Cook NB, Nordlund KV, Zwald NR, Rawson CL. 2006. Survey of management practices on reproductive performance of dairy cattle on large US commercial farms. J Dairy Sci, 89:4723-4735. Council on Dairy Cattle Breeding. 2014. Available on: http://www.cdcb.us. Accessed on: May, 2014. DeVries A, Olson JD, Pinedo PJ. 2010. Reproductive risk factors for culling and productive life in large dairy herds in the eastern United States between 2001 and 2006. J Dairy Sci, 93:613-623. Dechow C. 2014. Why Net Merit might be the wrong index for you. Hoard's Dairyman, 159:312. Fricke PM, Carvalho PD, Giordano JO, Valenza A, Lopes G, Amundson MC. 2014a. Expression and detection of estrus in dairy cows: the role of new technologies. Animal, 8(suppl. 1):134-143. Fricke PM, Giordano JO, Valenza A, Lopes G Jr, Amundson MC, Carvalho PD. 2014b. Reproductive performance of lactating dairy cows managed for first service using timed artificial insemination with or without detection of estrus using an activity-monitoring system. J Dairy Sci, 97:2771-2781. Galvão KN, Federico P, De Vries A, Schuenemann GM. 2013. Economic comparison of reproductive programs for dairy herds using estrus detection, timed artificial insemination, or a combination. J Dairy Sci, 96:2681-2693 Gifford CA, Racicot K, Clark DS, Austin KJ, Hansen TR, Lucy MC, Davies CJ, Ott TL. 2007. Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows. J Dairy Sci, 90:274-280. Gifford CA, Assiri AM, Satterfield MC, Spencer TE, Ott TL. 2008. Receptor transporter protein 4 (RTP4) in endometrium, ovary, and peripheral blood leukocytes of pregnant and cyclic ewes. Biol Reprod, 79:518-524. Goodling RC Jr, Shook GE, Weigel KA, Zwald NR. 2005. The effect of synchronization on genetic parameters of reproductive traits in dairy cattle. J Dairy Sci, 88:2217-2225. Green JA, Roberts RM. 2006. Establishment of an ELISA for the detection of native bovine pregnancyAnim. Reprod., v.11, n.3, p.207-216, Jul./Sept. 2014

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associated glycoproteins secreted by trophoblast binucleate cells. Methods Mol Med, 122:321-330. Green JC, Okamura CS, Poock SE, Lucy MC. 2010. Measurement of interferon-tau (IFN-tau) stimulated gene expression in blood leukocytes for pregnancy diagnosis within 18-20d after insemination in dairy cattle. Anim Reprod Sci, 12:24-33. Gumen A, Keskin A, Yilmazbas-Mecitoglu G, Karakaya E, Alkan A, Okut H, Wiltbank MC. 2012. Effect of presynchronization strategy before Ovsynch on fertility at first service in lactating dairy cows. Theriogenology, 78:1830-1838. Haller O, Kochs G. 2011. Human MxA protein: an interferon-induced dynamin-like GTPase with broad antiviral activity. J Interferon Cytokine Res, 31:79-87. Han H, Austin KJ, Rempel LA, Hansen TR. 2006. Low blood ISG15 mRNA and progesterone levels are predictive of non-pregnant dairy cows. J Endocrinol, 191:505-512. Herlihy MM, Giordano JO, Souza AH, Ayres H, Ferreira RM, Keskin A, Nascimento AB, Guenther JN, Gaska JM, Kacuba SJ, Crowe MA, Butler ST, Wiltbank MC. 2012. Presynchronization with DoubleOvsynch improves fertility at first postpartum artificial insemination in lactating dairy cows. J Dairy Sci, 95:7003-7014. Käppel ND, Pröll F, Gauglitz G. 2007. Development of a TIRF-based biosensor for sensitive detection of progesterone in bovine milk. Biosens Bioelectron, 22:2295-2300. Lima FS, De Vries A, Risco CA, Santos JE, Thatcher WW. 2010. Economic comparison of natural service and timed artificial insemination breeding programs in dairy cattle. J Dairy Sci, 93:4404-4413. Lucy MC, McDougall S, Nation DP. 2004. The use of hormonal treatments to improve the reproductive performance of lactating dairy cows in feedlot or pasture-based management systems. Anim Reprod Sci, 82/83:495-512. Miller RH, Norman HD, Kuhn MT, Clay JS, Hutchison JL. 2007. Voluntary waiting period and adoption of synchronized breeding in dairy herd improvement herds. J Dairy Sci, 90:1594-1606. Moore DA, Overton MW, Chebel RC, Truscott ML, BonDurant RH. 2005. Evaluation of factors that affect embryonic loss in dairy cattle. J Am Vet Med Assoc, 226:1112-1118. Moore K, Thatcher WW. 2006. Major advances associated with reproduction in dairy cattle. J Dairy Sci, 89:1254-1266. Moreira F, de la Sota RL, Diaz T, Thatcher WW. 2000. Effect of day of the estrous cycle at the initiation of a timed artificial insemination protocol on reproductive responses in dairy heifers. J Anim Sci, 78:1568-1576. Neves RC, Leslie KE, Walton JS, Leblanc SJ. 2012. Reproductive performance with an automated activity monitoring system versus a synchronized breeding Anim. Reprod., v.11, n.3, p.207-216, Jul./Sept. 2014

program. J Dairy Sci, 95:5683-5693. O’Connor ML. 1994. Alternative methods for determining pregnancy status. In: Proceedings of the National Reproduction Symposium, 1994, Pittsburgh, PA. Dallas, TX: Texas Agricultural Extension Service. pp. 23-32. Oliveira JF, Henkes LE, Ashley RL, Purcell SH, Smirnova NP, Veeramachaneni DN, Anthony RV, Hansen TR. 2008. Expression of interferon (IFN)stimulated genes in extrauterine tissues during early pregnancy in sheep is the consequence of endocrine IFN-tau release from the uterine vein. Endocrinology, 149:1252-1259. Ott TL, Yin J, Wiley AA, Kim HT, Gerami-Naini B, Spencer TE, Bartol FF, Burghardt RC, Bazer FW.1998. Effects of the estrous cycle and early pregnancy on uterine expression of Mx protein in sheep (Ovis aries). Biol Reprod, 59:784-794. Ott TL, Gifford CA. 2010. Effects of early conceptus signals on circulating immune cells: lessons from domestic ruminants. Am J Reprod Immunol, 64:245254. Pereira RV, Caixeta LS, Giordano JO, Guard CL, Bicalho RC. 2013. Reproductive performance of dairy cows resynchronized after pregnancy diagnosis at 31 (±3 days) after artificial insemination (AI) compared with resynchronization at 31 (±3 days) after AI with pregnancy diagnosis at 38 (±3 days) after AI. J Dairy Sci, 96:7630-7639. Pierson RA, Ginther OJ. 1984. Ultrasonography for detection of pregnancy and study of embryonic development in heifers. Theriogenology, 22:225-233. Pinedo PJ, DeVries A. 2010. Effect of days to conception in the previous lactation on the risk of death and live culling around calving. J Dairy Sci, 93:968977. Pontzer CH, Torres BA, Vallet JL, Bazer FW, Johnson HM. 1988. Antiviral activity of the pregnancy recognition hormone ovine trophoblast protein-1. Biochem Biophys Res Commun, 152:801-807. Pursley JR, Mee MO, Wiltbank MC. 1995. Synchronization of ovulation in dairy cows using PGF2alpha and GnRH. Theriogenology, 44:915-923. Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL. 1997. Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J Dairy Sci, 80:295-300. Quentela LA, Barrio M, Peña AI, Becerra JJ, Cainzos J, Herradón PG, Díaz C. 2012. Use of ultrasound in the reproductive management of dairy cattle. Reprod Domest Anim, 47(suppl. 3):34-44. Rabiee AR, Lean IJ, Stevenson MA. 2005. Efficacy of Ovsynch program on reproductive performance in dairy cattle: a meta-analysis. J Dairy Sci, 88:2754-2770. Ribeiro ES, Bruno RG, Farias AM, HernándezRivera JA, Gomes GC, Surjus R, Becker LF, Birt A, Ott TL, Branen JR, Sasser RG, Keisler DH, 215

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Thatcher WW, Bilby TR, Santos JE. 2014. Low doses of bovine somatotropin enhance conceptus development and fertility in lactating dairy cows. Biol Reprod, 90:112. Sasser RG, Ruder CA, Ivani KA, Butler JE, Hamilton WC. 1986. Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol Reprod, 35:936942. Schalue-Francis TK1, Farin PW, Cross JC, Keisler D, Roberts RM. 1991. Effect of injected bovine interferonalpha I1 on estrous cycle length and pregnancy success in sheep. J Reprod Fertil, 91:347-356. Souza AH, Ayres H, Ferreira RM, Wiltbank MC. 2008. A new presynchronization system (DoubleOvsynch) increases fertility at first postpartum timed AI in lactating dairy cows. Theriogenology, 70:208-215. Spencer TE, Bazer FW. 2004. Conceptus signals for establishment and maintenance of pregnancy. Reprod Biol Endocrinol, 2:49-64. Spencer TE, Johnson GA, Bazer FW, Burghardt RC. 2007. Fetal-maternal interactions during the establishment of pregnancy in ruminants. Soc Reprod Fertil Suppl, 64:379-396. Stevenson JL, Dalton JC, Ott TL, Racicot KE, Chebel RC. 2007. Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers. J Anim Sci, 85:21632172. Stevenson JS, Hill SL, Nebel RL, Dejarnette JM. 2014. Ovulation timing and conception risk after automated activity monitoring in lactating dairy cows. J

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Sex-sorted sperm for artificial insemination and embryo transfer programs in cattle M.F. Sá Filho1,5, M. Nichi1, J.G. Soares1, L.M. Vieira1, L.F Melo3, A. Ojeda2, E.P. Campos Filho4, A.H. Gameiro2, R. Sartori3, P.S. Baruselli1,5 1

Departamento de Reprodução-VRA, FMVZ-USP, São Paulo, SP, Brazil. Departamento de Nutrição e Produção Animal-VNP, FMVZ-USP, Pirassununga, SP, Brazil. 3 Departamento de Zootecnia, ESALQ-USP, Piracicaba-SP, Brazil. 4 Sexing Technologies, Sertãozinho, SP, Brazil.

2

Abstract The selection of offspring from the desired sex can be one of the determining factors to increase the genetic progress and farmer´s profitability in either beef or dairy cattle. In fact, the use of sex-sorted sperm has been applied worldwide combined with artificial insemination (AI) upon estrus detection in heifers. Additionally, several researches have been performed aiming to investigate the use of sex-sorted sperm during timed AI (TAI) programs and for insemination of superstimulated donors for in vivo embryo programs. Pregnancy per AI (P/AI) of cyclic heifers inseminated in estrus with sex-sorted sperm has been reported to be approximately 75 to 80% of the P/AI of heifers inseminated with non-sorted sperm. Insemination of superstimulated cows with sex-sorted sperm has been reported to reduce the production of viable embryos. Recently, however, it has been demonstrated that P/AI and embryo production per flushing resulting from AI with sex-sorted sperm may be improved when the time of AI is postponed in relation to the time of AI with non-sorted sperm. The P/AI of non stimulated females and fertilization rates and number of embryos recovered from superstimulated females were increased when AI occurred between 16 and 24 h after the onset of estrus (i.e. 6 to 14 h before ovulation). Nonetheless, despite the improvements achieved in the last decade, there is still a significant individual variability in fertility among bulls that have their sperm sex-sorted. It is critical that the pre-determination of the sire fertility is a paramount when sex-sorted sperm is utilized in commercial AI and ET programs. Thus, the aim of this review is to discuss the main concepts related to the use of sex-sorted sperm in TAI and ET programs, addressing some strategies to increase the efficiency of the technique. Keywords: bovine, fertility, reproduction, sexed semen, sorting technique. Introduction Sex-sorting of sperm cells by flow cytometry is an established method that has been commercially used _________________________________________ 5 Corresponding author: [email protected]; [email protected] Phone: +55(11)3091-7674; Fax: +55(11)3091-7412 Received: June 8, 2014 Accepted: July 13, 2014

in cattle (Seidel, 2007; Garner and Seidel, 2008; Rath et al., 2013). This technology is an important tool for the dairy and beef industry, leading to greater supply of replacement heifers and the consequent hastening on genetic gain (De Vries et al., 2008; Chebel et al., 2010). Specific in beef farms, the use of sex-sorted semen could increase the incidence of male calves, product of greater interest due to the increased meat production potential. The separation of sperm bearing X and Y chromosomes is possible due to the differences on the DNA content of these cells (X bearing sperm has about 4% more genetic material than Y bearing sperm) identified by flow cytometry (Johnson, 2000). The sex sorting process by flow cytometry is the most efficient method to separate X from Yspermatozoa in a large scale (Garner and Seidel, 2008; Rath et al., 2013; Seidel, 2014). Advances in semen sex sorting have enabled incorporation of this technology into commercial operations (De Vries et al., 2008; Norman et al., 2010). Despite the significant advances in sex-sorting sperm using flow cytometry in cattle, lower pregnancy per AI (P/AI) and reduced in vivo embryo production is achieved when compared to the rates obtained with non sex-sorted sperm (Schenk et al., 2006, 2009; Larson et al., 2010; Sales et al., 2011; Soares et al., 2011; Sá Filho et al., 2012; Seidel, 2014). The considerable interest in sex-sorting technology worldwide provides several research opportunities and challenges associated to the use of this product in farms. The aim of this review is to bring into focus a summary of our current understanding on the use of sex-sorted sperm in AI and ET programs, as well as strategies to optimize the efficiency of these combined technologies. Fertility after the use of sex-sorted sperm in cattle Despite the advances in sex-sorting of sperm using flow cytometry, lower P/AI is currently observed when compared with conventional semen (DeJarnette et al., 2009, 2010, 2011; Sales et al., 2011; Sá Filho et al., 2012). The P/AI of females inseminated with sex-sorted sperm may be influenced by their reduced lifespan in the uterus (Maxwell et al., 2004), reduced number of sorted sperm per straw (Seidel and Schenk, 2008;

Sá Filho et al. Sex-sorted sperm in cattle.

Schenk et al., 2009; DeJarnette et al., 2011) and bullrelated fertility (Frijters et al., 2009; Sá Filho et al., 2010a; DeJarnette et al., 2011; Sales et al., 2011). The reduced lifespan of the sex-sorted sperm in the female reproductive tract, due to mitochondria modification and DNA fragmentation, could alter the optimum interval to perform AI relative to ovulation (Maxwell et al., 2004; Sá Filho et al., 2010a; Gosálvez et al., 2011;Sales et al., 2011; Rath et al., 2013). In a combination of several experiments, Seidel et al. (1999) observed that the P/AI of heifers vary from 40 to 68% and from 67 to 82% in those females inseminated with sex-sorted and non-sex-sorted sperm, respectively.

Also, Seidel and Schenk (2008) observed a lower P/AI when using sex-sorted sperm (31 to 42%) than non sex-sorted sperm (43 to 62%). In zebu females (433 heifers and 230 non-suckling cows) inseminated with male-sexed sperm following estrus detection (Dominguez et al., 2011), lower P/AI was observed when AI was performed using sex-sorted sperm (38.8%; 131/338) than non sex-sorted sperm (57.9%; 188/325). Despite lower P/AI described in the literature in cattle inseminated using sex-sorted sperm; there is consensus that fertility of heifers inseminated upon estrous detection using sex-sorted sperm is about 70 to 80% of the P/AI obtained following the use of conventional semen (Table 1).

Table 1. Pregnancy per AI (P/AI) of females inseminated with non sex-sorted or sex-sorted sperm and the pregnancy proportion obtained by sex-sorted sperm based on non sex-sorted sperm. Pregnancy per AI based on type of semen used in AI Non sex-sorted % Sexed Proportion Breed Category Reference (n/n) % (n/n) % Timed Artificial insemination Beef Cows 54.2 (232/428) 45.4 (193/425) 83.7 Sá Filho et al., 2012 Beef Cows 54.7 (134/245) 45.9 (113/246) 83.9 Sá Filho et al., 2012 Beef Cows 51.8 (100/193) 41.8 (82/196) 80.7 Sales et al., 2011 Beef Cows 55.3 (105/190) 40.9 (79/193) 74.0 Sales et al., 2011 Souza et al., 2006, Dairy Cows 27.1 (44/162) 13.0 (21/161) 48.0 FMVZ/USP, São Paulo, Brazil, unpublished data Artificial insemination with estrus detection Beef Heifers 67.6 (96/142) Beef Heifers 67.0 (85/126) Beef Cows and Heifers 57.9 (188/325) Dairy Heifers 60.0 (1375/2292) Dairy Cows and Heifers 37.7 (160/426) 56.0 Dairy Heifers (30082/53718) Dairy Cows and Heifers 37.4 (34/91) Dairy Cows 46.0 (69/149) Dairy Heifers 60.0 (74/124) Dairy Heifers 62.0 (163/263) Overall

53.7 (130/242) 52.6 (129/245) 38.8 (131/338) 38.0 (881/2319) 22.9 (51/223)

79.4 78.5 67.0 63.3 60.7

Seidel and Schenk, 2008 Seidel and Schenk, 2008 Dominguez et al., 2011 DeJarnette et al., 2011 Mellado et al., 2010

45.0 (17893/39763)

80.3

DeJarnette et al., 2009

28.8 (38/132) 21.0 (33/157) 46.7 (114/244) 42.1 (225/534)

77.0 45.6 77.8 67.9

Bodmer et al., 2005 Andersson et al., 2006 Seidel and Schenk, 2008 Seidel and Schenk, 2008

44.3% (20113/45418)

79.1

56.0% (32941/58874)

In lactating dairy cows, a recent retrospective study demonstrated that the use of sex-sorted sperm for AI of US Holstein cows (10.8 million AI) was able to achieve mean P/AI about 25% (Norman et al., 2010). Andersson et al. (2006) reported that the average P/AI was 21% with sex-sorted sperm and 46% with non sex-sorted sperm in dairy cows. Schenk et al. (2009) verified that lactating dairy cows achieved ~25% of P/AI when using sex-sorted sperm and ~37% with non sex-sorted sperm. Other recent study (DeJarnette et al., 2010), evaluated the use of different doses of sex-sorted sperm and non sex-sorted sperm in lactating dairy cows. The P/AI of lactating cows were 23, 25, and 32% 218

following the use of 2.1 and 3.5 × 106 sex-sorted sperm dosages and 15 × 106 conventional, respectively. Also, in other recent study working with crossbred Bos indicus x Bos taurus lactating dairy cows, Sá Filho et al. (2013) reported lower P/AI in cows receiving AI using sex-sorted sperm following TAI (21.4%) than cows bred upon estrus detection (31.7%). In brief, the P/AI observed following the use of sex-sorted sperm is dependent on the P/AI normally observed following the use of conventional semen. Thus, similarly to what is observed when conventional semen is used, P/AI of females inseminated with sex-sorted semen is dependent on fertility of the bulls, Anim. Reprod., v.11, n.3, p.217-224, Jul./Sept. 2014

Sá Filho et al. Sex-sorted sperm in cattle.

animal categories (lactating cows or cyclic heifers), and management across different farms. Consequently, the major commercial recommendation for the use of sexsorted sperm still has been in heifers after detection of estrus, especially due to their higher fertility (DeJarnette et al., 2009; Norman et al., 2010; Healy et al., 2013). Improving the insemination

P/AI

by adjusting

the

time

of

The optimal time at which insemination should take place relative to ovulation depends primarily on the lifespan of spermatozoa and the viability of the oocyte in the female genital tract (Hunter and Wilmut, 1984). Dransfield et al. (1998) and Roelofs et al. (2006) demonstrated that the probability of P/AI decreased when AI using non sex-sorted sperm is performed closer to the moment of ovulation. According to Roelofs et al. (2006), fertilization drastically decreases when AI with conventional semen occurs after ovulation. Our research group performed a study to evaluate different times to perform AI using sex-sorted sperm. Thereby, Jersey heifers (n = 638) were inseminated following estrus detection using radio telemetry (Heat Watch®) in different intervals from onset of estrus to insemination (12 to 16 h; 16 to 20 h; 20 to 24 h and 24 to 30 h) . The P/AI of heifers inseminated from 12 to 16 h after the onset of estrus (37.7%; 40/106) was lower (P = 0.03) than those inseminated from 16.1 to 20 h (51.8%; 85/164) and 20.1 to 24 h (55.6%; 130/234). No differences were observed on P/AI for heifers inseminated from 24.1 to 30 h (45.5%; 61/134) when compared to the other interval groups. Therefore, increasing the interval from the onset of estrus to AI may increase pregnancy rates when using sex-sorted semen. This could be achieved by increasing the frequency of estrus detection or using methods that allow continuous monitoring of cow activity, e.g. mount monitoring systems. Furthermore, it is important to note that the effect of timing of insemination on pregnancy rate could be more pronounced when using sex-sorted sperm from bulls less tolerant to the sorting process. In Brazil, it has been recently reported that almost 60% of the AI performed in this country are made at fixed time (Baruselli et al., 2012). For this, the use of a P4/progestin plus E2 based TAI protocols has been the most commercially used type of fixed-time synchronization protocol (Baruselli et al., 2012). In these ovulation synchronization protocols, an intravaginal device containing P4 or an ear implant containing norgestomet and estradiol benzoate (EB; 2mg i.m.) are administered on day 0; an injection of prostaglandin (PG) F2α on day 8 or 9 at the moment of device withdrawal plus 300 to 400 IU of equine chorionic gonadotropin (eCG). Different ovulation

Anim. Reprod., v.11, n.3, p.217-224, Jul./Sept. 2014

inducers with similar efficiency could be used such as estradiol cipionate (EC; 0.5 mg i.m.) at moment or EB (1mg i.m.) 24 h after the P4/progestin implant removal. Timed artificial insemination usually is performed from 48 to 54 h after P4/progestin source removal (Baruselli et al., 2012). A possibility to improve P/AI following the use of sex-sorted sperm is to control the variation in the time of ovulation through the use of ovulation synchronization protocols. For instance, in beef and dairy females, P4/E2 based synchronization protocols induce ovulation 70-72 h after the P4 device removal (Souza et al., 2009; Sales et al., 2011; Baruselli et al., 2012). Because sex-sorted sperm presents lower viability on the reproductive tract than conventional semen (Maxwell et al., 2004), our research group has evaluated P/AI following delayed AI using sex-sorted sperm in heifers. In a first study, Sales et al. (2011) inseminated 420 cyclic Jersey heifers at either 54 or 60 h after P4 device removal, using either sex-sorted (2.1 million of sperm) or non sex-sorted sperm (20 million of sperm) from three sires. The interaction between time of AI and type of semen tended (P = 0.06) to affect P/AI. Delayed insemination improved P/AI only when sex-sorted sperm was used (TAI 54 h = 16.2%; 17/105 vs. TAI 60 h = 31.4%; 32/102). In contrast, altering the timing of AI did not affect P/AI with non sex-sorted sperm (TAI 54 h = 50.5%; 51/101 vs.TAI 60 h = 51.8%; 58/112). Based on these results, Sales et al. (2011) used the same experimental design in suckled Bos indicus cows. Timing of AI did not improve P/AI of cows receiving sex-sorted semen and the interaction between time of AI and type of semen did not affect P/AI [Non sex-sorted TAI 54 h = 48.4% (n = 95) vs. Non sexsorted TAI 60 h = 55.1% (n = 98) and Sex-sorted TAI 54 h = 37.4% (n = 99) vs. Sex-sorted TAI 60 h = 46.4% (n = 97)]. Finally, the same authors evaluated the moment of insemination using sex-sorted sperm relative to the moment of ovulation in suckled Bos indicus cows (n = 339). In this study, cows were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ovarian ultrasound examinations were performed twice daily in all cows to verify the moment of ovulation. Ovulation occurred, on average, 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. Higher P/AI (37.9%, 36/95) was observed for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly lower for TAI performed between 12.1 and 24 h (19.4%, 21/108) or >24 h (5.8%, 5/87) before ovulation. Therefore, improvement on P/AI with delayed time of AI is possible (Table 2), and seemed achievable when breeding is performed 60 h after progestin implant removal compared with the standard 54 h normally used in TAI protocols.

219

Sá Filho et al. Sex-sorted sperm in cattle.

Table 2. Influence of the AI moment, diameter of the largest follicle (LF) and presence of corpus luteum (CL) on the pregnancy rate of heifers and cows submitted to synchronization of ovulation protocols. Pregnancy per AI % (n/n) Reference Animal category Early AI time Late AI time P value Schenk et al. (2009) Angus heifers 34.4 (11/32) 48.6 (17/35) >0.10 Neves (2010) Nelore cows 20.8 (27/130) 30.9 (38/123) 0.05). At 24 and 48 hours of cooling, the 1+1 dilution resulted in lower progressive motility (PM) (P < 0.001) at 15°C (22.5±9.11, 11.66±7.66) and 5°C (15.83±11.66, 4.77±7.13). At 24 hours, progressive motility at 5 or 15°C was similar among the dilutions 1+2 (36.66±12.36, 42.77±14.26) and 1+3 (42.5 ± 11.53; 44.72±11.94). At 48 hours, PM was higher (P 0.05) total and progressive motilities. Therefore, if variables had an effect, it was due to SP presence. Motile and progressive spermatozoa increased (P0.05) the variables. Thus, addition of 10% SP after thawing improves semen quality. Acknowledgements: FAPESP Process 2011/23484-8 and 2013/08070-8 and Botupharma®.

Anim. Reprod., v.11, n.3, p.299, Jul./Sept. 2014

299

Proceedings of the 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE), August 14 to 17th, 2014, Natal, RN, Brazil. Abstracts.

A020 Male Reproductive Physiology and Semen Technology

Cellrox deep red® for the detection of oxidative stress in ram sperm by in vitro induction M.B.R. Alves, E.C.C. Celeghini, A.F.C. Andrade, R.P. Arruda, L. Batissaco, T.G. Almeida FMVZ-USP.

Keywords: fluorescent probe, reactive oxygen species, semen. The sperm cell is highly susceptible to damage caused by oxidative stress (OS). It is one of the factors that are associated with male infertility. Therefore, it is very important to find new analyses methods that are easier to perform and read. The CellROX Deep Red Reagent® fluorescent probe (CAT 10422, Life Technologies, New York, USA) presents a number of advantages, but there are no published studies of its use on sperm. Thus, the aim of the present study is to evaluate the effectiveness of this method in samples of ram semen induced to in vitro OS. The analyses were conducted in four ejaculates of three White Dorper rams (n=12) that were between 14 and 24 months of age. Semen was collected by artificial vagina. The ejaculates were treated in T0, T50 and T100. T0 corresponded to the control semen sample that was not submitted to OS induction, T50 corresponded to the semen sample content 50% without OS induction and 50% inducted to OS, and T100 corresponded to the semen sample that was entire submitted to OS induction. The OS induction was performed by adding iron sulfate (4 mM) and ascorbic acid (20 mM). For the preparation of the technique, 200 µL (25x106 spermatozoa/mL) of each semen sample (T0, T50 and T100) was used and 0.5 µL of CellROX® (1 mM) and 2 µL of Hoechst 33342 (2.5 mg/mL, Life Technologies) were added. The semen sample was incubated at 37° C/30 minutes. After incubation, the sample was centrifuged at 2,000g/5 minutes, the supernatant was removed and the pellet was resuspended in 200 µL of TALP sperm. The cells were classified as sperm under mild or no OS (unstained midpiece), sperm under moderate OS (midpiece stained pale red), and sperm under intense OS (midpiece stained strong red). Data obtained from T0, T50 and T100 treatments were evaluated by analysis of variance (ANOVA) and the means were compared using Fisher's LSD test. OS data in T0, T50 and T100 treatments were subjected to linear regression analysis by Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). The experimental model used was: Y = a + bX, wherein Y is the estimative of OS due to treatment, a is the linear regression coefficient corresponding to the value of Y when X is 0, b is the regression coefficient for the percent of X on the response Y and X is the treatment. A significant difference (p